Stable modulators of gamma-c-cytokine activity

ABSTRACT

Disclosed herein are stable peptide antagonists based on the consensus yc-subunit binding site to inhibit the activity of yc-cytokines. Such peptide antagonists are capable of inhibiting the activity of multiple yc-cytokine family members. The yc-family cytokines are associated with important human diseases, such as leukemia, autoimmune diseases, collagen diseases, diabetes mellitus, skin diseases, degenerative neuronal diseases and graft-versus-host disease (GvHD). Thus, inhibitors of yc-cytokine activity are valuable therapeutic and cosmetic agents as well as research tools.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/483,210, filed on Apr. 7, 2017, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING IN ELECTRONIC FORMAT

The present application is being filed along with an Electronic SequenceListing as an ASCII text file via EFS-Web. The Electronic SequenceListing is provided as a file entitled BION010WOSEQLIST.txt, created andlast saved on Mar. 30, 2018, which is 60,939 bytes in size. Theinformation in the Electronic Sequence Listing is incorporated herein byreference in its entirety in accordance with 35 U.S.C. § 1.52(e).

BACKGROUND Field

Some embodiments relate to peptide antagonists of γc-family cytokines, agroup of mammalian cytokines that are mainly produced by epithelial,stromal and immune cells and control the normal and pathologicalactivation of a diverse array of lymphocytes. Some embodiments alsorelate to the therapeutic uses of such peptides for the treatment ofcertain human diseases. The present embodiments also relate to thecosmeceutical applications of such peptides. Description of targetdiseases, cosmeceutical applications, as well as methods ofadministration, production, and commercialization of the peptides aredisclosed.

Description of the Related Art

Cytokines are a diverse group of soluble factors that mediate variouscell functions, such as, growth, functional differentiation, andpromotion or prevention of programmed cell death (apoptotic cell death).Cytokines, unlike hormones, are not produced by specialized glandulartissues, but can be produced by a wide variety of cell types, such asepithelial, stromal or immune cells.

The γc-family cytokines are a group of mammalian cytokines that aremainly produced by epithelial, stromal and immune cells and control thenormal and pathological activation of a diverse array of lymphocytes.These cytokines are critically required for the early development of Tcells in the thymus as well as their homeostasis in the periphery.

SUMMARY

In some embodiments, a composite peptide comprising amino acid sequencesof at least two interleukin (IL) protein gamma-c-box D-helix regions,wherein the composite peptide comprises an amino acid sequenceD/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2), wherein X denotes anyamino acid, wherein the composite peptide comprises at least twoalpha-alkenyl substituted amino acids, and wherein the at least twoalpha-alkenyl substituted amino acids are linked via at least oneintra-peptide hydrocarbon linker element is provided.

In some embodiments of the composite peptide, the at least twoalpha-alkenyl substituted amino acids are linked to form the at leastone intra-peptide hydrocarbon linker element by ring closing metathesis,wherein the ring closing metathesis is catalyzed by Grubb's catalyst.

In some embodiments, an amino acid in the composite peptide is selectedfrom the group consisting of natural amino acids, non-natural aminoacids, (D) stereochemical configuration amino acids, (L) stereochemicalconfiguration amino acids, (R) stereochemical configuration amino acidsand (S) stereochemical configuration amino acids, and wherein the atleast two alpha-alkenyl substituted amino acids are selected from thegroup consisting of R-propenylalanine (CAS: 288617-76-5; R3Ala),S-propenylalanine (CAS: 288617-71-0; S3Ala), D-allylglycine (CAS:170642-28-1; D3Gly), L-allylglycine (CAS: 146549-21-5; L3Gly),R-pentenylalanine (CAS: 288617-77-6; R5Ala), S-pentenylalanine (CAS:288617-73-2; S5Ala), R-pentenylglycine (CAS: 1093645-21-6; R5Gly),S-pentenylglycine (CAS: 856412-22-1; S5Gly), R-butenylalanine (CAS:1311933-82-0; R4Ala), S-butenylalanine (CAS: 288617-72-1; S4Ala),R-butenylglycine (CAS: 865352-21-2; R4Gly), S-butenylglycine (CAS:851909-08-5; S4Gly), R-hexenylalanine (CAS: 288617-78-7; R6Ala),S-hexenylalanine (CAS: 288617-74-3; S6Ala), R-hexenylglycine (CAS:1208226-88-3; R6Gly), S-hexenylglycine (CAS: 1251904-51-4; S6Gly),R-heptenylalanine (CAS: 1311933-84-2; R7Ala), S-heptenylalanine (CAS:1311933-83-1; S7Ala), R-heptenylglycine (CAS: 1262886-63-4; R7Gly),S-heptenylglycine (CAS: 1058705-57-9; S7Gly), di-substitutedbis-propenylglycine (CAS: 1311992-97-8; bis3Gly), di-substitutedbis-pentenylglycine (CAS: 1068435-19-7; bis5Gly), di-substitutedbis-butenylglycine (bis4Gly), di-substituted bis-hexenylglycine(bis6Gly), di-substituted bis-heptenylglycine (bis7Gly),R-octenylalanine (CAS: 945212-26-0; R8Ala), S-octenylalanine (CAS:288617-75-4; S8Ala), R-octenylglycine (CAS: 1191429-20-5; R8Gly), andS-octenylglycine (CAS: 1262886-64-5; S8Gly).

In some embodiments of the composite peptide, the at least twoalpha-alkenyl substituted amino acids linked by the at least oneintra-peptide hydrocarbon are separated by n−2 amino acids, wherein nrepresents the number of amino acids encompassed by the intra-peptidelinkage.

In some embodiments of the composite peptide, when the at least twoalpha-alkenyl substituted amino acids linked by the at least oneintra-peptide hydrocarbon are separated by three amino acids, the atleast one intra-peptide hydrocarbon linker element spans a singleα-helical turn of the composite peptide.

In some embodiments of the composite peptide, when the composite peptidecomprises one or more non-contiguous single α-helical turns, the aminoacid positions that correlate with a single α-helical turn of thecomposite peptide correspond to amino acid positions i and i+4 of thecomposite peptide, where i is the first amino acid position of thesingle α-helical turn and i+4 is the last amino acid position of thesingle a-helical turn, and wherein amino acid positions i and i+4comprise alpha-alkenyl substituted amino acids.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R-propenylalanine (CAS: 288617-76-5; R3Ala),S-propenylalanine (CAS: 288617-71-0; S3Ala), D-allylglycine (CAS:170642-28-1; D3Gly), and L-allylglycine (CAS: 146549-21-5; L3Gly), andthe alpha-alkenyl substituted amino acid at position i+4 is selectedfrom the group consisting of R-pentenylalanine (CAS: 288617-77-6;R5Ala), S-pentenylalanine (CAS: 288617-73-2; S5Ala), R-pentenylglycine(CAS: 1093645-21-6; R5Gly), and S-pentenylglycine (CAS: 856412-22-1;S5Gly), the hydrocarbon linker element formed by the ring-closingmetathesis is represented by Formula 1.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, and the alpha-alkenylsubstituted amino acid at position i+4 is selected from the groupconsisting of R3Ala, S3Ala, D3Gly, and L3Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula2.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R-butenylalanine (CAS: 1311933-82-0; R4Ala),S-butenylalanine (CAS: 288617-72-1; S4Ala), R-butenylglycine (CAS:865352-21-2; R4Gly), and S-butenylglycine (CAS: 851909-08-5; S4Gly), andthe alpha-alkenyl substituted amino acid at position i+4 is selectedfrom the group consisting of R4Ala, S4Ala, R4Gly, and S4Gly, thehydrocarbon linker element formed by the ring-closing metathesis isrepresented by Formula 3.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, and the alpha-alkenylsubstituted amino acid at position i+4 is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula4.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R4Ala, S4Ala, R4Gly, and S4Gly, and the alpha-alkenylsubstituted amino acid at position i+4 is selected from the groupconsisting of R-hexenylalanine (CAS: 288617-78-7; R6Ala),S-hexenylalanine (CAS: 288617-74-3; S6Ala), R-hexenylglycine (CAS:1208226-88-3; R6Gly), and S-hexenylglycine (CAS: 1251904-51-4; S6Gly),the hydrocarbon linker element formed by the ring-closing metathesis isrepresented by Formula 5.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, and S6Gly, and the alpha-alkenylsubstituted amino acid at position i+4 is selected from the groupconsisting of R4Ala, S4Ala, R4Gly, and S4Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula6.

In some embodiments of the composite peptide, the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R3Ala, S3Ala, D3Gly, and L3Gly, and the alpha-alkenylsubstituted amino acid at position i+4 is selected from the groupconsisting of R-heptenylalanine (CAS: 1311933-84-2; R7Ala),S-heptenylalanine (CAS: 1311933-83-1; S7Ala), R-heptenylglycine (CAS:1262886-63-4; R7Gly), and S-heptenylglycine (CAS: 1058705-57-9; S7Gly),the hydrocarbon linker element formed by the ring-closing metathesis isrepresented by Formula 7.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, and the alpha-alkenylsubstituted amino acid at position i+4 is selected from the groupconsisting of R3Ala, S3Ala, D3Gly, and L3Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula8.

In some embodiments of the composite peptide, when the composite peptidecomprises two or more contiguous single α-helical turns, the amino acidpositions that correlate with the first single α-helical turn of thecomposite peptide correspond to amino acid positions i and i+4 of thecomposite peptide, where i is the first amino acid position of the firstsingle α-helical turn and i+4 is the last amino acid position of thefirst single a-helical turn, and the amino acid positions that correlatewith the second single α-helical turn of the composite peptidecorrespond to amino acid positions i+4 and i+8 of the composite peptide,where i+4 is the first amino acid position of the second singleα-helical turn and i+8 is the last amino acid position of the secondsingle α-helical turn, and wherein amino acid positions i, i+4 and i+8comprise alpha-alkenyl substituted amino acids.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, the alpha-alkenylsubstituted amino acid at position i+4 is di-substitutedbis-propenylglycine (CAS: 1311992-97-8; bis3Gly), and the alpha-alkenylsubstituted amino acid at position i+8 is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula9.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R3Ala, S3Ala, D3Gly, and L3Gly, the alpha-alkenylsubstituted amino acid at position i+4 is di-substitutedbis-pentenylglycine (CAS: 1068435-19-7; bis5Gly), and the alpha-alkenylsubstituted amino acid at position i+8 is selected from the groupconsisting of R3Ala, S3Ala, D3Gly, and L3Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula10.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R4Ala, S4Ala, R4Gly, and S4Gly, the alpha-alkenylsubstituted amino acid at position i+4 is di-substitutedbis-butenylglycine (bis4Gly), and the alpha-alkenyl substituted aminoacid at position i+8 is selected from the group consisting of R4Ala,S4Ala, R4Gly, and S4Gly, the hydrocarbon linker element formed by thering-closing metathesis is represented by Formula 11.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, the alpha-alkenylsubstituted amino acid at position i+4 is bis5Gly, and the alpha-alkenylsubstituted amino acid at position i+8 is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula12.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, and S6Gly, the alpha-alkenylsubstituted amino acid at position i+4 is bis4Gly, and the alpha-alkenylsubstituted amino acid at position i+8 is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, or S6Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula13.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R4Ala, S4Ala, R4Gly, and S4Gly, the alpha-alkenylsubstituted amino acid at position i+4 is di-substitutedbis-hexenylglycine (bis6Gly), and the alpha-alkenyl substituted aminoacid at position i+8 is selected from the group consisting of R4Ala,S4Ala, R4Gly, and S4Gly, the hydrocarbon linker element formed by thering-closing metathesis is represented by Formula 14.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, the alpha-alkenylsubstituted amino acid at position i+4 is bis3Gly, and the alpha-alkenylsubstituted amino acid at position i+8 is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula15.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R3Ala, S3Ala, D3Gly, and L3Gly, the alpha-alkenylsubstituted amino acid at position i+4 is di-substitutedbis-heptenylglycine (bis7Gly), and the alpha-alkenyl substituted aminoacid at position i+8 is selected from the group consisting of R3Ala,S3Ala, D3Gly, and L3Gly, the hydrocarbon linker element formed by thering-closing metathesis is represented by Formula 16.

In some embodiments of the composite peptide, when the at least twoalpha-alkenyl substituted amino acids linked by the at least oneintra-peptide hydrocarbon are separated by six residues, the at leastone intra-peptide hydrocarbon linker element spans a double α-helicalturn of the composite peptide.

In some embodiments of the composite peptide, when the composite peptidecomprises one or more non-contiguous double α-helical turns, the aminoacid positions that correlate with a double α-helical turn of thecomposite peptide correspond to amino acid positions i and i+7 of thecomposite peptide, where i is the first amino acid position of thedouble α-helical turn and i+7 is the last amino acid position of thedouble a-helical turn, and wherein amino acid positions i and i+7comprise alpha-alkenyl substituted amino acids.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, and the alpha-alkenylsubstituted amino acid at position i+7 is selected from the groupconsisting of R-octenylalanine (CAS: 945212-26-0; R8Ala),S-octenylalanine (CAS: 288617-75-4; S8Ala), R-octenylglycine (CAS:1191429-20-5; R8Gly), and S-octenylglycine (CAS: 1262886-64-5; S8Gly),the hydrocarbon linker element formed by the ring-closing metathesis isrepresented by Formula 17.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R8Ala, S8Ala, R8Gly, and S8Gly, and the alpha-alkenylsubstituted amino acid at position i+7 is selected from the groupconsisting of R5Ala, S5Ala, R5Gly, and S5Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula18.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, and S6Gly, and the alpha-alkenylsubstituted amino acid at position i+7 is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula19.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, and the alpha-alkenylsubstituted amino acid at position i+7 is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, and S6Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula20.

In some embodiments of the composite peptide, when the composite peptidecomprises two or more contiguous double α-helical turns, the amino acidpositions that correlate with the first double α-helical turn of thecomposite peptide correspond to amino acid positions i and i+7 of thecomposite peptide, where i is the first amino acid position of the firstdouble α-helical turn and i+7 is the last amino acid position of thefirst double α-helical turn, and the amino acid positions that correlatewith the second double a-helical turn of the composite peptidecorrespond to amino acid positions i+7 and i+14 of the compositepeptide, where i+7 is the first amino acid position of the second doubleα-helical turn and i+14 is the last amino acid position of the seconddouble α-helical turn, and wherein amino acid positions i, i+7 and i+14comprise alpha-alkenyl substituted amino acids.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R8Ala, S8Ala, R8Gly, and S8Gly, the alpha-alkenylsubstituted amino acid at position i+7 is bis5Gly, and the alpha-alkenylsubstituted amino acid at position i+14 is selected from the groupconsisting of R8Ala, S8Ala, R8Gly, and S8Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula21.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, the alpha-alkenylsubstituted amino acid at position i+7 is bis6Gly, and the alpha-alkenylsubstituted amino acid at position i+14 is selected from the groupconsisting of R7Ala, S7Ala, R7Gly, and S7Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula22.

In some embodiments of the composite peptide, when the alpha-alkenylsubstituted amino acid at position i is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, and S6Gly, the alpha-alkenylsubstituted amino acid at position i+7 is bis7Gly, and the alpha-alkenylsubstituted amino acid at position i+14 is selected from the groupconsisting of R6Ala, S6Ala, R6Gly, and S6Gly, the hydrocarbon linkerelement formed by the ring-closing metathesis is represented by Formula23.

In some embodiments, a derivative of the composite peptide comprises anamino acid sequence that shares about 50% to about 99% identity with thecomposite peptide.

In some embodiments, the composite peptide inhibits the activity of oneor more γc-cytokines selected from the group consisting of IL-2, IL-4,IL-7, IL-9, IL-15, and IL-21.

In some embodiments, the composite peptide further comprises a signalpeptide. In some embodiments, the composite peptide further comprisesone or more additional moieties conjugated at the N-terminus, C-terminusor a side residue of the composite peptide. In some embodiments, thecomposite peptide further comprises the one or more additional moietiesare selected from the group consisting of bovine serum albumin (BSA),albumin, Keyhole Limpet Hemocyanin (KLH), Fc region of IgG, a biologicalprotein that functions as scaffold, an antibody against a cell-specificantigen, a receptor, a ligand, a metal ion, and Poly Ethylene Glycol(PEG).

In some embodiments, the composite peptide comprises amino acidsequences of at least two interleukin (IL) protein gamma-c-box D-helixregions, wherein the composite peptide comprises the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), and wherein thecomposite peptide comprises at least two alpha-alkenyl substituted aminoacids, and wherein the at least two alpha-alkenyl substituted aminoacids are linked via at least one intra-peptide hydrocarbon linkerelement.

In some embodiments, the composite peptide comprises amino acidsequences of at least two interleukin (IL) protein gamma-c-box D-helixregions, wherein the composite peptide comprises the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), and whereinthe composite peptide comprises at least two alpha-alkenyl substitutedamino acids, and wherein the at least two alpha-alkenyl substitutedamino acids are linked via at least one intra-peptide hydrocarbon linkerelement.

In some embodiments, the composite peptide the composite peptideinhibits a cell growth promoting activity of IL-15, IL-21, or acombination thereof. In some embodiments, the composite peptide inhibitsa STAT5 signaling activity of IL-15. In some embodiments, the compositepeptide inhibits a STAT3 signaling activity of IL-21.

In some embodiments, a pharmaceutical composition comprising atherapeutically effective amount of a composite peptide, or a derivativethereof, and a pharmaceutically acceptable carrier, diluent, excipientor combination thereof is provided. In some embodiments of thepharmaceutical composition, the composite peptide is selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Insome embodiments of the pharmaceutical composition, the compositepeptide or the derivative thereof modulates the activity of one or moreγc-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21.

In some embodiments, a method of preventing or treating aγc-cytokine-mediated disease is provided. In some embodiments, themethod comprises administering to a subject in need thereof, apharmaceutical composition comprising a therapeutically effective amountof a composite peptide, or a derivative thereof, and a pharmaceuticallyacceptable carrier, diluent, excipient or combination thereof, whereinthe composite peptide is selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein the composite peptidecomprises at least two alpha-alkenyl substituted amino acids, andwherein the at least two alpha-alkenyl substituted amino acids arelinked via at least one intra-peptide hydrocarbon linker element,wherein the composite peptide or derivative thereof modulates theactivity of one or more γc-cytokines selected from the group consistingof IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the derivativethereof comprises an amino acid sequence that shares about 50% to about99% identity with the composite peptide, thereby preventing or treatingthe γc-cytokine-mediated disease.

In some embodiments, the γc-cytokine-mediated disease is selected fromthe group consisting of CD4-leukemia, CD8-leukemia, LGL-leukemia,systemic lupus erythematosis, Sjögren's syndrome, Wegener'sgranulomatosis, Celiac disease, Hashimoto's thyroiditis, rheumatoidarthritis, diabetes mellitus, psoriasis, multiple sclerosis, uvietis,inflammation of the eye, graft-versus-host disease (GvHD), inflammatorybowel diseases (IBD, including ulcerative colitis and Crohn's disease),Systemic Lupus Erythematosus, and alopecia areata.

In some embodiments, a method of preventing or treating anHTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP)associated disease is provided. In some embodiments, the methodcomprises administering to a subject in need thereof, a pharmaceuticalcomposition comprising a therapeutically effective amount of a compositepeptide, or a derivative thereof, and a pharmaceutically acceptablecarrier, diluent, excipient or combination thereof, wherein thecomposite peptide is selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, and SEQ ID NO: 3, wherein the composite peptide comprisesat least two alpha-alkenyl substituted amino acids, and wherein the atleast two alpha-alkenyl substituted amino acids are linked via at leastone intra-peptide hydrocarbon linker element, wherein the compositepeptide or the derivative thereof modulates the activity of one or moreγc-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21, wherein the derivative thereof comprises anamino acid sequence that shares about 50% to about 99% identity with thecomposite peptide, thereby preventing or treating an HTLV-1-associatedmyelopathy (HAM)/tropical spastic paraparesis (TSP) associated disease.

In some embodiments, the HAM/TSP associated disease is selected from thegroup consisting of Adult T-cell Leukemia (ATL), HTLV-associatedMyelopathy/Tropical Spastic Paraparesis (HAM/TSP), and othernon-neeoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy, andmyositis.

In some embodiments, a method of preventing or treating an inflammatoryrespiratory disease is provided. In some embodiments, the methodcomprises administering to a subject in need thereof, a pharmaceuticalcomposition comprising a therapeutically effective amount of a compositepeptide, or a derivative thereof, and a pharmaceutically acceptablecarrier, diluent, excipient or combination thereof, wherein thecomposite peptide is selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, and SEQ ID NO: 3, wherein the composite peptide comprisesat least two alpha-alkenyl substituted amino acids, and wherein the atleast two alpha-alkenyl substituted amino acids are linked via at leastone intra-peptide hydrocarbon linker element, wherein the compositepeptide or the derivative thereof modulates the activity of one or moreγc-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21, wherein the derivative thereof comprises anamino acid sequence that shares about 50% to about 99% identity with thecomposite peptide, thereby preventing or treating an inflammatoryrespiratory disease.

In some embodiments, the inflammatory respiratory disease is selectedfrom the group consisting of asthma, sinusitis, hay fever, bronchitis,chronic obstructive pulmonary disease (COPD), allergic rhinitis, acuteand chronic otitis, and lung fibrosis.

In some embodiments, a method of preventing or treating a cosmeticcondition is provided. In some embodiments, the method comprisesadministering to a subject in need thereof, a pharmaceutical compositioncomprising a therapeutically effective amount of a composite peptide, ora derivative thereof, and a pharmaceutically acceptable carrier,diluent, excipient or combination thereof, wherein the composite peptideis selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO: 3, wherein the composite peptide comprises at least twoalpha-alkenyl substituted amino acids, and wherein the at least twoalpha-alkenyl substituted amino acids are linked via at least oneintra-peptide hydrocarbon linker element, wherein the composite peptideor the derivative thereof modulates the activity of one or moreγc-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21, wherein the derivative thereof comprises anamino acid sequence that shares about 50% to about 99% identity with thecomposite peptide, thereby preventing or treating a cosmetic condition.

In some embodiments, the cosmetic condition is selected from the groupconsisting of acne, hair loss, sunburn, nail maintenance, and appearanceof aging.

In some embodiments, a kit for preventing or treating a condition in apatient is provided. In some embodiments of the kit, the condition is aγc cytokine-mediated disease, an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease, aninflammatory respiratory disease, a cosmetic condition, or a combinationthereof. In some embodiments, the kit comprises a pharmaceuticalcomposition, wherein the pharmaceutical composition comprising atherapeutically effective amount of a composite peptide or a derivativethereof, and a pharmaceutically acceptable carrier, diluent, excipientor combination thereof, wherein the composite peptide is selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3,wherein the composite peptide comprises at least two alpha-alkenylsubstituted amino acids, and wherein the at least two alpha-alkenylsubstituted amino acids are linked via at least one intra-peptidehydrocarbon linker element, wherein the composite peptide or thederivative thereof modulates the activity of one or more γc-cytokinesselected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, andIL-21, and wherein the derivative thereof comprises an amino acidsequence that shares about 50% to about 99% identity with the compositepeptide.

In some embodiments of the kit, the condition is one or more of CD4leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosus,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, rheumatoid arthritis, diabetes mellitus,psoriasis, multiple sclerosis, uveitis, inflammation of the eye,graft-versus-host disease (GvHD), inflammatory bowel diseases (IBD,including ulcerative colitis and Crohn's disease), Systemic LupusErythematosus, alopecia areata, Adult T-cell Leukemia (ATL),HTLV-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), andother non-neeoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy,myositis, asthma, sinusitis, hay fever, bronchitis, chronic obstructivepulmonary disease (COPD), allergic rhinitis, acute and chronic otitis,and lung fibrosis, acne, hair loss, sunburn, nail maintenance, orappearance of aging.

In some embodiments of the composite peptide, the one or morecarbon-carbon double bonds present in the intra-peptide hydrocarbonlinker are utilized for one or more organic chemical reactions to addone or more additional chemical functionalities. In some embodiments ofthe composite peptide, the one or more organic chemical reactionscomprises an alkene reaction. In some embodiments of the compositepeptide, the alkene reaction is selected from the group consisting ofhydroboration, oxymercuration, hydration, chlorination, bromination,addition of HF, HBr, HCl or HI, dihydroxylation, epoxidation,hydrogenation, and cyclopropanation. In some embodiments of thecomposite peptide, one or more additional chemical functionalities canbe added subsequent to the alkene reaction wherein the one or moreadditional chemical functionalities comprise a covalent addition of oneor more chemical group substituents, wherein the covalent addition ofone or more chemical group substituents comprises nucleophilic reactionswith epoxide and hydroxyl groups. In some embodiments of the compositepeptide, the one or more additional chemical functionalities areselected from the group consisting of biotin, radioisotopes, therapeuticagents, rapamycin, vinblastine, taxol, non-protein fluorescent chemicalgroups, FITC, hydrazide, rhodamine, maleimide, protein fluorescentgroups, GFP, YFP, and mCherry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an alignment of the D-helix region of human γc-cytokinefamily members.

FIG. 1B depicts the γc-box (SEQ ID NO: 10) and IL-2/IL-15 box (SEQ IDNO: 11) motifs which give rise to the consensus sequence around theD-helix region of the γc-cytokines.

FIG. 2 depicts a diagramed representation of the biochemical propertiesof amino acids.

FIG. 3A shows inhibition of IL-15 and IL-9 activity by BNZ-γ in a PT-18proliferation assay.

FIG. 3B shows a proliferation assay of CTLL-2 cells grown in thepresence of IL-2 or IL-15 and 0, 0.1, 1 or 10 μM BNZ-γ.

FIG. 3C shows inhibition of IL-15 and IL-21 activity by SEQ ID NO: 3 ina NK92 proliferation assay.

FIG. 3D shows a proliferation assay of NK92 cells grown in the presenceof IL-2, IL-15, or IL-21 and a μM dose response of SEQ ID NO: 3.

FIG. 4A shows inhibition of IL-15-mediated tyrosine-phosphorylation ofSTAT5 by BNZ-γ.

FIG. 4B shows inhibition of IL-15-mediated phosphorylation of STAT5, butnot IL-2-mediated phosphorylation of STAT5 in CTLL-2 cells by SEQ ID NO:3, and inhibition of IL-21-mediated phosphorylation of STAT3 in NK92cells by SEQ ID NO: 3.

FIG. 5A shows an ex vivo T-cell proliferation assay using HAM/TSPperipheral blood. T-cell proliferation is inhibited by addition ofBNZ-γ.

FIG. 5B shows the population of CD4+CD25+ cells in an ex vivo T-cellproliferation assay using HAM/TSP peripheral blood is diminished afteradding BNZ-γ to the culture.

FIG. 5C shows the population of CD4+Ki67 cells in an ex vivo T-cellproliferation assay using HAM/TSP peripheral blood is reduced afteradding BNZ-γ to the culture.

FIG. 5D shows the percent of live cells by Guava staining in an ex vivoT-cell proliferation assay using HAM/TSP peripheral blood is notimpacted after adding BNZ-γ to the culture.

FIG. 6 shows the alignment of the sequence of SEQ ID NO: 3 to theD-helix regions of different human γc-cytokine family members. Theshaded areas represent amino acid sequences of the human γc-cytokinefamily members that are identical to their corresponding amino acids inthe sequence of SEQ ID NO: 3.

FIG. 7 shows the relative inhibition of IL-15 activity by a series ofcustom peptide derivatives of SEQ ID NO: 3 containing an intra-peptidehydrocarbon linker element spanning a single helical turn (amino acidpositions i and i+4) or two helical turns (amino acid positions i andi+7) in the alpha-helical secondary structure of each custom peptidederivative as compared to the unmodified SEQ ID NO: 3 in a NK92proliferation assay.

FIG. 8A shows a time-course protease stability measurement of unmodifiedSEQ ID NO: 3 in comparison with a representative custom peptidederivative of SEQ ID NO: 3 containing one hydrocarbon linker element(SEQ ID NO: 39), and another representative custom peptide derivative ofSEQ ID NO: 3 containing two hydrocarbon linker elements and certainamino acid positions in the (D) stereochemical configuration (SEQ ID NO:57) in simulated intestinal fluid over 60 minutes.

FIG. 8B shows a time-course protease stability measurement of arepresentative custom peptide derivative of SEQ ID NO: 3 containing onehydrocarbon linker element (SEQ ID NO: 39) in comparison with arepresentative custom peptide derivative of SEQ ID NO: 3 containing twohydrocarbon linker elements (SEQ ID NO: 83) in simulated intestinalfluid over 120 minutes.

FIG. 9 shows inhibition of IL-15 and IL-21 induced gene transcription ofinterferon gamma (IFNg) by SEQ ID NO: 83 (10 μM), but not that of thenon-γc cytokine IL-12 induction of IFNg gene transcription in the humanNK92 cell line.

DETAILED DESCRIPTION Overview

More than 100 cytokines have been identified so far and are consideredto have developed by means of gene duplications from a pool ofprimordial genes (See Bazan, J. F. 1990, Immunol. Today 11:350-354). Insupport of this view, it is common for a group of cytokines to share acomponent in their multi-subunit receptor system. The mostwell-documented shared cytokine subunit in T cells is the common γsubunit (γc-subunit).

The γc-subunit is shared by 6 known cytokines (Interleukin-2 (IL-2),Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin-9 (IL-9),Interleukin-15 (IL-15), and Interleukin-21 (IL-21), collectively calledthe “γc-cytokines” or “γc-family cytokines”) and plays an indispensablerole in transducing cell activation signals for all these cytokines.Additionally, for each of the γc-cytokines, there are one or two privatecytokine-specific receptor subunits that when complexed with theγc-subunit, give rise to a fully functional receptor. (See Rochman etal., 2009, Nat Rev Immunol. 9: 480-90.)

The γc-family cytokines are a group of mammalian cytokines that aremainly produced by epithelial, stromal and immune cells and control thenormal and pathological activation of a diverse array of lymphocytes.These cytokines are critically required for the early development of Tcells in the thymus as well as their homeostasis in the periphery. Forexample, in the absence of the γc-subunit, T, B and NK cells do notdevelop in mice. (See Sugamura et al., 1996, Annu. Rev. Immunol.14:179-205).

The γc-cytokines are important players in the development of thelymphoid cells that constitute the immune system, particularly T, B, andNK cells. Further, γc-cytokines have been implicated in various humandiseases. Thus, factors that inhibit γc-cytokine activity would provideuseful tools to elucidate the developmental mechanism of subsets oflymphocytes and to treat immune disorders and γc-cytokine-mediateddiseases.

Germ line depletion of the genes encoding the γc-subunit in mice ormutations of γc-subunit in humans are known to cause severe combinedimmunodeficiency (SCID) by disrupting the normal appearance or functionof NK, T, and B cells. The importance of the γc-subunit in the signaltransduction of the γc-cytokines, IL-2, -4, -7, -9, 15,-21, is indicatedin studies demonstrating the lack of response of lymphocytes from thesemice and human patients to the γc-cytokines (reviewed in Sugamura etal., 1995 Adv. Immunol. 59:225-277). This indicates that disruption ofthe interaction between the γc-subunit and a γc-cytokine wouldefficiently block the intracellular signaling events by the γc-cytokinefamily members. Therefore antagonist peptides according to the presentembodiments are expected to effectively block the pathogenic changes inhumans suffering from the diseases mediated by misregulation of theγc-cytokine family members.

As an alternative to antibody-mediated approaches for modulating theactivity of individual γc-cytokines, Applicants have devised novel, lowmolecular weight compounds herein referred to as “Simul-Block”, whichsuppress the activity of multiple γc-cytokines. These low molecularweight compounds, which include both chemicals and peptides, are lessimmunogenic than antibodies. These properties distinguish Simul-Block asa more efficient strategy for mediating γc-cytokine activity in clinicalinterventions.

Pathologies Associated with the γc-Cytokines

Recent studies have indicated that dysregulation of expression anddysfunction of the γc-cytokines could lead to a wide variety of humanimmunologic and hematopoietic diseases.

IL-2

While IL-2 was historically considered a prototype T cell growth factor,the generation of a knockout mouse lacking IL-2 expression revealed thatIL-2 is not critical for the growth or development of conventional Tcells in vivo. Over-expression of IL-2, however, leads to a preferentialexpansion of a subset of T-cells; the regulatory T cells (T-regs). (SeeAntony et al., 2006, J. Immunol. 176:5255-66.) T-regs suppress theimmune responses of other cells and thus act to maintain peripheraltolerance (reviewed in Sakaguchi et al., 2008, Cell 133:775-87).Breakdown of peripheral tolerance is thought to cause autoimmunediseases in humans.

Thus, the immunosuppressive function of T-regs is thought to prevent thedevelopment of autoimmune diseases (See Sakaguchi et al., 2008, Cell133:775-87). T-regs have also been implicated in cancer, where solidtumors and hematologic malignancies have been associated with elevatednumbers of T-regs (See De Rezende et al., 2010, Arch. Immunol. Ther.Exp. 58:179-190).

IL-4

IL-4 is a non-redundant cytokine involved in the differentiation of Thelper cells into the Th2 (T-helper type 2) subset, which promotes thedifferentiation of premature B cells into IgE-producing plasma cells.IgE levels are elevated in allergic asthma. Thus, IL-4 is implicated inthe development of allergic Asthma. Antibodies targeting IL-4 can beused to treat or even prevent the onset of allergic asthma. (See LeBuanec et al., 2007, Vaccine 25:7206-16.)

IL-7

IL-7 is essential for B cell development and the early development of Tcells in the thymus. In mice, the abnormal expression of IL-7 causesT-cell-associated leukemia. (See Fisher et al., 1993, Leukemia2:S66-68.) However, in humans, misregulation of IL-7 does not appear tocause T-cell-associated leukemia. In humans, up-regulation of IL-7either alone or in combination with another γc-cytokine family member,IL-15, has been implicated in Large Granular Lymphocyte (LGL) leukemia.

IL-9

The role of IL-9 is still rather uncharacterized compared to otherγc-cytokine family members. Mice depleted of the IL-9 gene appear normaland do not lack any subsets of cells in the lymphoid and hematopoieticcompartments. Recent studies, however, reveal an in vivo role for IL-9in the generation of Th17 (T-helper induced by interleukin-17) cells(See Littman et al., 2010, Cell 140(6):845-58; and Nowak et al., 2009,J. Exp. Med. 206: 1653-60).

IL-15

IL-15 is critically involved in the development of NK cells, NK-T cells,some subsets of intraepithelial lymphocytes (IELs), γδ-T cells, andmemory-phenotype CD8 T-cells (See Waldmann, 2007, J. Clin. Immunol.27:1-18; and Tagaya et al., 1996, EMBO J. 15:4928-39.) Over-expressionof IL-15 in mice leads to the development of NK-T cell and CD8 cell typeT cell leukemia (See Fehniger et al., 2001, J. Exp. Med. 193:219-31;Sato et al. 2011 Blood 117:4032-40). These experimentally inducedleukemias appear similar to LGL (large-granular lymphocyte) leukemia inhumans, since in both instances the leukemic cells express CD8 antigen.

It is also suspected that IL-15-mediated autocrine mechanisms may beinvolved in the leukemic transformation of CD4 T lymphocytes. (See Azimiet al., 1998, Proc. Natl. Acad. Sci. 95:2452-7; Azimi et al., 1999, J.Immunol. 163:4064-72; Azimi et al., 2000, AIDS Res. Hum. Retroviruses16:1717-22; and Azimi et al., 2001, Proc. Natl. Acad. Sci. 98:14559-64).For example, CD4-tropic HTLV-I, which causes Adult T cell leukemia inhumans, induces autocrine growth of virus-transformed T cells throughthe production of IL-15 and IL-15Rα (Azimi et al., 1998, Proc. Natl.Acad. Sci. 95:2452-7).

In addition to leukemic transformation, recent studies implicate IL-15in the pathological development of Celiac disease (CD), an autoimmunedisease. IL-15 is known to stimulate the differentiation of NK, CD8 andintestinal intraepithelial lymphocyte (IEL) cells intolymphokine-activated killer (LAK) cells by inducing the expression ofcytolytic enzymes (i.e., Granzyme and Perforin) as well as interferon-y.Celiac Disease (denoted CD from herein) is an immune-mediatedenteropathy that is triggered by the consumption of gluten-containingfood in individuals that express specific HLA-DQ alleles.

The prevalence of this disease is 1% in the western population. The onlycurrent treatment for CD is the complete elimination of gluten from thepatient's diet. The pathology of CD is mainly caused by extensive damageto the intestinal mucosa, which is caused by activated CD8 T cells thathave infiltrated to the intestinal lamina propria. These CD8 T cellsappear to be activated through mechanisms involving IL-15. One recentpublication demonstrated in mice that ectopic over-expression of IL-15by enterocytes leads to the development of enteropathy, which closelyresembles the lesions in CD patients. Neutralization of IL-15 activitydramatically diminished the pathological changes. Thus, an interventionblocking the activation of CD8 T cells by IL-15 appears to provide analternative strategy in managing CD to the conventional gluten-freediet.

IL-21

IL-21 is the most recently discovered member of the γc-family. Unlikeother family members, IL-21 does not appear to have potentgrowth-promoting effects. Instead, IL-21 is thought to function more asa differentiation factor than a factor controlling cellularproliferation (See Tagaya, 2010, J. Leuk. Biol. 87:13-15).

Current Strategies for Treating γc-Cytokine-Mediated Disorders

As γc-cytokines are thought to be involved in numerous human diseases,several methods of treating γc-cytokine-implicated diseases byinhibiting γc-cytokine family activities have been proposed. Thesemethods include the use of cytokine-specific monoclonal antibodies toneutralize the targeted cytokine's activity in vivo; use of monoclonalantibodies targeting the private cytokine-specific receptor subunits(subunits other than the shared γc-subunit) to selectively inhibitcytokine activity; and use of chemical inhibitors that block thedownstream intracellular cytokine signal transduction pathway.

While cytokine-specific antibodies are often the first choice indesigning therapeutics, cytokines that share receptor components displayoverlapping functions (See Paul, W. E., 1989, Cell 57:521-24) and morethan one cytokine can co-operate to cause a disease (See Examplesdescribed herein). Thus, approaches involving neutralization of a singlecytokine may not be effective in the treatment of cytokine-implicatedhuman diseases.

Strategies for designing therapeutics that inhibit the function ofmultiple cytokines via antibodies which recognize a shared receptorcomponent have also been proposed. However, the multi-subunit nature ofcytokine receptor systems and the fact that functional receptors for asingle cytokine can assume different configurations makes this approachdifficult.

For example, a functional IL-15 receptor can be either IL-15Rβ/γc orIL-15Rα/β/γc. (See Dubois et al., 2002, Immunity 17:537-47.) An antibodyagainst the IL-15Rβ receptor (TMβ1), is an efficient inhibitor of theIL-15 function, but only when the IL-15Ru molecule is absent from thereceptor complex. (See Tanaka et al., 1991, J. Immunol. 147:2222-28.)Thus, the effectiveness of a monoclonal anti-receptor antibody, whetherraised against a shared or a private subunit, can be context-dependentand is unpredictable in vivo.

Although clinical use of monoclonal antibodies against biologicallyactive factors or receptors associated with the pathogenesis of diseasesis an established practice, there are few demonstrations of successfuloutcomes. Moreover, establishment of a clinically-suited monoclonalantibody treatment is a long and difficult process, with the successfulgeneration of a neutralizing antibody largely a matter of luck. Forexample, due to the critical importance of the γc-subunit in mediatingsignaling by γc-family cytokines, many attempts to generate polyclonaland monoclonal antibodies against the γc-subunit have been made andthere exist many commercial antibodies recognizing the γc-subunit inmice and in humans. Curiously, however, none of these anti-γc-subunitantibodies block the function of the γc-cytokines.

Another problem with the therapeutic use of monoclonal antibodies isthat monoclonal antibodies are usually generated by immunizing rodentswith human proteins, so the generated antibody is a foreign protein andthus highly immunogenic. To circumvent this problem, the amino acidsequence of the monoclonal antibody is molecularly modified so that theantibody molecule is recognized as a human immunoglobulin (a processcalled humanization), but this process requires time and expense.

Targeting JAK3, as an Existing Alternative Example for the Inhibition ofMultiple γc-Cytokines

The interaction between the γc-subunit and a γc-cytokine leads to theactivation of an intracellular protein tyrosine kinase called Januskinase 3 (Jak3). Jak3, in turn, phosphorylates multiple signalingmolecules including STAT5, and PI3 kinase. The interaction of theγc-subunit and Jak3 is very specific. In fact, there is no otherreceptor molecule that recruits Jak3 for signal transduction. (SeeO'Shea, 2004, Ann. Rheum. Dis. 63:(suppl. II):ii67-7.) Thus, theinhibition of cytokine signaling through the γc-subunit can beaccomplished by blocking the activity of Jak3 kinase. Accordingly,multiple chemical inhibitors that target the kinase activity of Jak3have been introduced to the market. (See Pesu et al., 2008, Immunol.Rev. 223:132-142.) One such example is CP690,550.

The major shortcoming of these protein kinase inhibitors is the lack ofspecificity to Jak3 kinase. These drugs intercept the binding of ATP(adenosine-triphosphate) molecules to Jak3 kinase, a common biochemicalreaction for many protein kinases, and thus tend to block the action ofmultiple intracellular protein kinases that are unrelated to Jak3 kinasewhose actions are critically needed for the well-being of normal cellsin various tissues. Thus, more specific inhibitors of signaling throughthe γc-subunit are needed.

There is therefore a great need for an alternative strategy for treatingγc-cytokine-implicated diseases.

Discovery of the γc-Box

The C-terminus (the D-helix) of the γc-cytokines contains the proposedsite for interacting with the common γc-subunit of the multi-unitcytokine receptors. (Bernard et al., 2004 J. Biol. Chem. 279:24313-21.)Comparison of the biochemical properties of the amino acids of allγc-cytokines identified in mice and humans revealed that the chemicalnature of the amino acids, for example, hydrophobicity, hydrophilicity,base/acidic nature, are conserved, if not identical, at many positionsin the D-helix across the members of the γc-cytokine family.

In contrast, the sequence of IL-13, which is related to the γc-cytokineIL-4, but does not bind to the γc-subunit, does not exhibit significanthomology in the D-helix region to the γc-cytokines, suggesting that thesequence homology in the D-helix region is correlated with binding tothe γc-subunit. As shown in FIG. 1A, alignment of the amino acidsequences of the D-helix region of γc-cytokine family members in humansreveals a motif of moderate sequence homology in these cytokinesreferred to herein as “the γc-box”.

The γc-box (SEQ ID NO: 10) comprises 19 amino acids where out of the 19positions, positions 4, 5, and 13 are fully conserved as Phenylalanine,Leucine, and Glutamine, respectively. Less conservation is observed atpositions 6, 7 and 11 of the γc-box where the amino acid is one of twoor three related amino acids that share physico-chemical properties:position 6 may be occupied by the polar amino acids Glutamate,Asparagine or Glutamine; non-polar amino acids Serine or Arginine canoccupy position 7; and position 11 is occupied by either of thenon-polar aliphatic amino acids Leucine or Isoleucine. Positions 9 and16 may be occupied by the either the non-polar amino acid Isoleucine orthe polar amino acid Lysine. See FIG. 1B. Some differences in the aminoacid composition of the γc-box are observed at positions 9 and 16amongst subfamilies of the γc-cytokines. Comparison of the γc-cytokinesacross species indicates that Isoleucine is often present at the 9 and16 positions in the IL-2/15 subfamily, whereas the other γc-familymembers often possess Lysine in these positions. Not wishing to be boundby a particular theory, Isoleucine and Lysine are biochemicallydifferent and thus may impart specific conformational differencesbetween the IL-2/15 subfamily and other γc-cytokines.

Conservation of the γc-box motif between γc-cytokines is supported byfindings that a Glutamine (Gln, Q) residue located in the D-helix regionis critical for the binding of the γc-cytokines to the γc-subunit.(Bernard et al., 2004 J. Biol. Chem. 279: 24313-21.)

Peptide Inhibitors of γc-Cytokine Activity

The activity of γc-family cytokines may be blocked by disrupting theinteraction between the γc-cytokine and the γc-subunit, for example byintroducing a competitive inhibitor which can interact with theγc-subunit without stimulating signaling through the multi-subunitcytokine receptors. Not to be bound by a particular theory, theconserved γc-box motif, which participates in binding of the γc-familycytokines to the γc-subunit, presents a core base amino acid sequencewhich can be utilized to design peptide inhibitors of γc-cytokinesignaling.

Provided herein are stable composite peptides, and compositions, kitsand/or systems thereof to modulate γc-cytokine signaling. The terms“composite peptide,” “oligopeptide,” “polypeptide,” “peptide,” and“protein” can be used interchangeably when referring to the “custompeptide derivatives” provided in accordance with the present embodimentsand can be used to designate a series of amino acid residues of anylength. The peptides of the present embodiments may be linear or cyclic.The peptides of the present embodiments may include natural amino acids,non-natural amino acids amino acids in (D) stereochemical configuration,amino acids in (L) stereochemical configuration, amino acids in (R)stereochemical configuration, amino acids in (S) stereochemicalconfiguration, or a combination thereof.

The core γc-box amino acid sequence comprises:D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2) (where X denotes anyamino acid). At least some embodiments described herein relate to custompeptide derivatives of the core γc-box amino acid sequence which caninhibit the activity of one or more γc-cytokines. Custom peptidederivatives include any peptide whose partial amino acid sequence showsapproximately 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%or 99.8% identity to the core γc-box amino acid sequence. Custom peptidederivatives further include any peptide wherein a partial amino acidsequence of that peptide derivative comprises amino acids with similarphysico-chemical properties to the amino acids of the core γc-box. Forexample, amino acids with similar physico-chemical properties wouldinclude Phenylalanine, Tyrosine, Tryptophan, and Histidine, which arearomatic amino acids. FIG. 2 shows a diagrammed representation of aminoacids with similar physico-chemical properties which may be may besubstituted for the amino acids comprising the core γc-box. Peptidederivatives of the core γc-box may be 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25-30, 30-35, 35-40, 40-45, 45-50, or more than50 amino acids in length. In some embodiments, the custom peptidederivatives may be conjugated to the N-termini, C-termini and/or to theside residues of existing biological proteins/peptides.

Some embodiments described herein relate to enhancing the formation ofand/or stabilizing one or more helical secondary structures of custompeptide derivatives of the core γc-box. In some embodiments, a helicalsecondary structure of a custom peptide derivative of the core γc-box isenhanced and/or stabilized by incorporating one or more intra-peptidehydrocarbon linker elements (often referred to as “staples” in theliterature).

As used herein, the term “staples,” “hydrocarbon,” “hydrocarbonlinker-element,” “hydrocarbon linker chain,” “linker element,” “linkerchain” or “linker” refers to any carbon-containing chemical chain(straight or branched) comprising at least 1 to about 20 carbon atoms.The carbon-containing chemical chain may have one or more chemical groupsubstituents covalently attached within the chain. The carbon-containingchemical chain may have one or more carbon-carbon double bonds in eitherZ or E geometric configurations, or a combination of Z and E geometricconfigurations. The carbon-containing chemical chain may also have oneor more carbon-carbon triple bonds. In some embodiments, thecarbon-containing chemical chain may have one or more carbon-carbondouble bonds in either Z or E geometric configurations, or a combinationof Z and E geometric configurations and may additionally have one ormore carbon-carbon triple bonds. The carbon-containing chemical chainmay also covalently incorporate halogen atom substitutions (one or moreof fluorine, chlorine, bromine, and/or iodine) according to the chemicalvalences allowed by the halogen atom. The carbon containing chemicalchain may covalently incorporate heteroatom substitutions (one or moreof oxygen, nitrogen, and/or sulfur) according to the chemical valencesallowed by the heteroatom. The carbon-containing chemical chain maycovalently incorporate, without limitations, one or more carbon aromaticring systems (non-limiting examples include phenyl, naphthyl, anthracyl,etc.) with or without substituents, one or more heteroaryl ring systems(non-limiting examples include furan, thiophene, pyrrole, pyridine,pyran, oxazine, thiazine, pyrimidine, pyridazine, pyrazine, thiine,pyrazole, imidazole, triazole, indole, quinolone, isoxazole, oxazole,isothiazole, thiazole, etc.) with or without substituents, one or morenon-aromatic carbon ring systems (non-limiting examples includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.) with or withoutsubstituents, and one or more heterocyclyl ring systems (non-limitingexamples include ethylene oxide, ethylene imine, ethylene sulfide,tetrahydrofuran, tetrahydrothiophene, tetrahydropyran,tetrahydrothiopyran, dioxane, pyrrolidine, piperidine, piperazine,morpholine, etc.) with our without substituents. Suitable substituents,without limitation, include one or more of alkyl, alkaryl, aryl,aralkyl, alkoxy, thioalkoxy, aryloxy, haloalkyl, halo, oxo, nitro,hydroxy, mercapto, carboxy, alkylcarbonyl, alkoxycarbonyl,alkanesulfonyl, amino, amido, azido, cyano, PEG, affinity labels,targeting moiety, fatty-acid derived acyl group, biotin, radioisotopes,therapeutic agents (non-limiting examples include rapamycin,vinblastine, taxol, etc.), non-protein fluorescent chemical groups(non-limiting examples include FITC, hydrazide, rhodamine, maleimide,etc.), and protein fluorescent groups (non-limiting examples includeGFP, YFP, mCherry, etc.).

In some embodiments, one or more linker elements can join any two ormore separate custom peptide derivatives of the present disclosure. Insome embodiments, when one or more linker elements join any two or moreseparate custom peptide derivatives, the linker elements are referred toas inter-peptide linker elements.

In some embodiments, core γc-box custom peptide derivatives comprise twoor more α-alkenyl substituted amino acids. In some embodiments, the twoor more a-alkenyl substituted amino acids are linked via one or moreintra-peptide hydrocarbon linker elements incorporated at the α-alkenylsubstituted amino acids. In some embodiments, the a-alkenyl substitutedamino acids are utilized to catalyze the formation of an intra-peptidehydrocarbon linker element by ring-closing metathesis during peptidesynthesis. Intra-peptide linker elements join separate amino acids onthe same sequence of a custom peptide derivative of the presentdisclosure. In some embodiments, the peptides of the present disclosureare linear or cyclic. In some embodiments, one or more peptides have acombination of inter-peptide and intra-peptide hydrocarbon linkers. Itwill be appreciated by one of ordinary skill in the art that anycombination of linear and cyclic peptides and any combination and numberof inter-peptide and intra-peptide hydrocarbon linkers are possible. Forexample, a circular peptide may have one or more intra-peptide linkerslinking two substituted amino acids (e.g., α-alkenyl substituted aminoacids) within the circular peptide. The circular peptide may additionalbe linked via one or more inter-peptide linkers to another peptidecomprising one or more substituted amino acids (e.g., α-alkenylsubstituted amino acids).

Non-limiting examples of α-alkenyl substituted amino acids includeR-propenylalanine (CAS: 288617-76-5; R3Ala), S-propenylalanine (CAS:288617-71-0; S3Ala), D-allylglycine (CAS: 170642-28-1; D3Gly),L-allylglycine (CAS: 146549-21-5; L3Gly), R-pentenylalanine (CAS:288617-77-6; R5Ala), S-pentenylalanine (CAS: 288617-73-2; S5Ala),R-pentenylglycine (CAS: 1093645-21-6; R5Gly), S-pentenylglycine (CAS:856412-22-1; S5Gly), R-butenylalanine (CAS: 1311933-82-0; R4Ala),S-butenylalanine (CAS: 288617-72-1; S4Ala), R-butenylglycine (CAS:865352-21-2; R4Gly), S-butenylglycine (CAS: 851909-08-5; S4Gly),R-hexenylalanine (CAS: 288617-78-7; R6Ala), S-hexenylalanine (CAS:288617-74-3; S6Ala), R-hexenylglycine (CAS: 1208226-88-3; R6Gly),S-hexenylglycine (CAS: 1251904-51-4; S6Gly), R-heptenylalanine (CAS:1311933-84-2; R7Ala), S-heptenylalanine (CAS: 1311933-83-1; S7Ala),R-heptenylglycine (CAS: 1262886-63-4; R7Gly), S-heptenylglycine (CAS:1058705-57-9; S7Gly), di-substituted bis-propenylglycine (CAS:1311992-97-8; bis3Gly), di-substituted bis-pentenylglycine (CAS:1068435-19-7; bis5Gly), di-substituted bis-butenylglycine (bis4Gly),di-substituted bis-hexenylglycine (bis6Gly), di-substitutedbis-heptenylglycine (bis7Gly), R-octenylalanine (CAS: 945212-26-0;R8Ala), S-octenylalanine (CAS: 288617-75-4; S8Ala), R-octenylglycine(CAS: 1191429-20-5; R8Gly), and S-octenylglycine (CAS: 1262886-64-5;S8Gly) (See TABLE 1). Other amino acid substitutions are alsocontemplated and within the scope of this disclosure.

It will be understood by one of ordinary skill in the art that any aminoacid at any of the positions in the embodiments of the peptidesdisclosed herein can be a substituted amino acid (e.g., an α-alkenylsubstituted amino acid). In some embodiments, other modifications ofamino acids that allow for the formation of one or more inter-peptideand/or intra-peptide linkages (e.g., via hydrocarbon linker elements)are also contemplated.

Intra-peptide hydrocarbon linker elements have been shown to increasestability of peptide(s) by decreasing the susceptibility of thepeptide(s) to proteolytic digestion (reviewed in Walensky and Bird,2014, J. Med. Chem. 57:6275-88, which is hereby incorporated byreference in its entirety). Thus, in some embodiments, intra-peptidehydrocarbon linker elements decrease the susceptibility of peptides todegradation by serine proteases, cysteine proteases, threonineproteases, aspartic proteases, glutamic proteases, metalloproteases,asparagine peptide lyases or a combination thereof. In some embodiments,intra-peptide hydrocarbon linker elements improve the biologicalactivity of certain peptides derived from alpha-helical regions bystabilizing the bioactive secondary structure of peptide. In someembodiments, a biological activity corresponds to inhibition of theeffects of a cytokine.

Based on the identification of the conserved γc-box motif in cytokineswhich bind to the γc-subunit, Applicants have devised a novel, 19-mercustom derivative peptide which is an artificial composite peptidecombining partial amino acid sequences of both the human IL-2 and IL-15γc-box. The 19-mer peptide, herein referred to as BNZ-γ, consists of theamino acid sequence: I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), where the amino acids depicted by bold characters are conservedbetween IL-2 and IL-15 and the underlined amino acids representpositions where the physico-chemical properties of the amino acids areconserved.

Applicants discovered that the 19-mer BNZ-γ, suppresses IL-15 and IL-9induced cellular proliferation, but not IL-3 or IL-4 induced cellularproliferation. See FIG. 3A and EXAMPLE 2. Applicants furtherdemonstrated that BNZ-γ inhibits IL-15 mediated phosphorylation of theintracellular cytokine signal transduction molecule, STAT-5. See FIG. 4Aand EXAMPLE 5. These results demonstrate that custom peptide derivativesof the conserved γc-box motif can inhibit the activity of multipleγc-cytokines.

Several embodiments relate to custom derivative peptides of the 19-merBNZ-γ amino acid sequence, I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ IDNO: 1), which can inhibit the activity of one or more γc-cytokines.Custom peptide derivatives of the 19-mer BNZ-γ amino acid sequenceinclude any peptide whose partial amino acid sequence showsapproximately 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99%or 99.8% identity to amino acid sequence:I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). Custom peptidederivatives further include any peptide wherein a partial amino acidsequence of that peptide derivative comprises amino acids with similarphysico-chemical properties to the amino acids of sequence:I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). In severalembodiments, the amino acid residues of the custom derivative peptidesretain similar physico-chemical properties with the amino acid residuesof BNZ-γ, but exhibit different biological inhibition specificity to the6 γc-cytokine family members from that of the original 19-mer peptide.Peptide derivatives of BNZ-γ may be 19, 20, 21, 22, 23, 24, 25-30,30-35, 35-40, 40-45, 45-50, or more than 50 amino acids in length. Insome embodiments, the custom peptide derivatives may be conjugated tothe N-termini, C-termini and/or to the side residues of existingbiological proteins/peptides. The other moieties may include proteins orpeptides that stabilize the composite peptide, or other moieties,including without limitation, bovine serum albumin (BSA), albumin,Keyhole Limpet Hemocyanin (KLH), Fc region of IgG, a biological proteinthat functions as scaffold, an antibody against a cell-specific antigen,a receptor, a ligand, a metal ion and Poly Ethylene Glycol (PEG).

In some embodiments, any of the custom peptide derivatives disclosedherein can comprise one or more intra-peptide hydrocarbon linkerelements. In some embodiments, the 19-mer BNZ-γ (SEQ ID NO: 1) comprisesone or more intra-peptide hydrocarbon linker elements. In someembodiments, the 19-mer BNZ-γ (SEQ ID NO: 1) comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 4 residues apart on SEQ ID NO: 1. In someembodiments, the 19-mer BNZ-γ (SEQ ID NO: 1) comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 7 residues apart on SEQ ID NO: 1. In someembodiments, the 19-mer BNZ-γ (SEQ ID NO: 1) comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 4 residues apart on SEQ ID NO: 1 and 7 residuesapart on SEQ ID NO: 1.

In some embodiments, when the 19-mer BNZ-γ (SEQ ID NO: 1) is part of alonger peptide sequence, none of the amino acids joined through eachintra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position N terminal to the first residue of SEQ ID NO: 1. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Nterminal to the first residue of SEQ ID NO: 1. In some embodiments, 0,1, 2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position N terminal tothe first residue of SEQ ID NO: 1.

In some embodiments, when the 19-mer BNZ-γ (SEQ ID NO: 1) is part of alonger peptide sequence, none of the amino acids joined through eachintra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position C terminal to the last residue of SEQ ID NO: 1. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Cterminal to the last residue of SEQ ID NO: 1. In some embodiments, 0, 1,2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position C terminal tothe last residue of SEQ ID NO: 1.

In some embodiments, when the 19-mer BNZ-γ (SEQ ID NO: 1) is part of alonger peptide sequence, none of the amino acids joined through eachintra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position N terminal to the first residue of SEQ ID NO: 1 and at aposition C terminal to the last residue SEQ ID NO: 1. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Nterminal to the first residue of SEQ ID NO: 1 and at a single position Cterminal to the last residue of SEQ ID NO: 1. In some embodiments, 0, 1,2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position N terminal tothe first residue of SEQ ID NO: 1 and at a position C terminal to thelast residue of SEQ ID NO: 1.

In some embodiments of the 19-mer BNZ-γ (SEQ ID NO: 1), none or one ormore of the amino acids are joined through each intra-peptidehydrocarbon linker element positioned on the one or more additionalmoieties conjugated to one or more of the residues of SEQ ID NO: 1. Insome embodiments, 0 or 1 of the amino acids joined through eachintra-peptide hydrocarbon linker element may be positioned on the one ormore additional moieties conjugated to SEQ ID NO: 1. In someembodiments, 0, 1, 2, 3, 4 or 5 of the amino acids joined through eachintra-peptide hydrocarbon linker element may be positioned on the one ormore additional moieties conjugated to SEQ ID NO: 1.

In some embodiments of the 19-mer BNZ-γ, 0, 1, or 2 of the amino acidsjoined through each intra-peptide hydrocarbon linker element may includenatural or non-natural amino acids in the (D) as well as (L), or the (R)as well as (S), stereochemical configuration. In some embodiments of the19-mer BNZ-γ, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acidsjoined through each intra-peptide hydrocarbon linker element comprisenatural amino acids, non-natural amino acids, or a combination thereof.In some embodiments of the 19-mer BNZ-γ, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 of the amino acids joined through each intra-peptide hydrocarbonlinker element comprise the (D) stereochemical configuration, the (L)stereochemical configuration, or a combination thereof. In someembodiments of the 19-mer BNZ-γ, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ofthe amino acids joined through each intra-peptide hydrocarbon linkerelement comprise the (R) stereochemical configuration, the (S)stereochemical configuration, or a combination thereof.

In some embodiments of the 19-mer BNZ-γ, 0, 1, or 2 of the amino acidsjoined through each intra-peptide hydrocarbon linker element areconnected through a substituted side chain alpha carbon ornon-substituted side chain alpha carbon. In some embodiments of the19-mer BNZ-γ, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the amino acidsjoined through each intra-peptide hydrocarbon linker element areconnected through a substituted side chain alpha carbon, anon-substituted side chain alpha carbon or a combination thereof. Theγc-box amino acid sequence comprising D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q(SEQ ID NO: 2) (where X denotes any amino acid) is the conservedsequence identified in the D-helix of each member of the γc-cytokinefamily that is important for interacting with the common γc-subunit ofeach multi-unit cytokine receptor.

In some embodiments, any of the custom peptide derivatives disclosedherein can comprise one or more intra-peptide hydrocarbon linkerelements. In some embodiments, the composite peptide of SEQ ID NO: 2comprises one or more intra-peptide hydrocarbon linker elements. In someembodiments, the composite peptide of SEQ ID NO: 2 comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 4 residues apart on SEQ ID NO: 2. In someembodiments, the composite peptide of SEQ ID NO: 2 comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 7 residues apart on SEQ ID NO: 2. In someembodiments, the composite peptide of SEQ ID NO: 2 comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 4 residues apart on SEQ ID NO: 2 and 7 residuesapart on SEQ ID NO: 2.

In some embodiments, when the composite peptide of SEQ ID NO: 2 is partof a longer peptide sequence, none of the amino acids joined througheach intra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position N terminal to the first residue of SEQ ID NO: 2. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Nterminal to the first residue of SEQ ID NO: 2. In some embodiments, 0,1, 2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position N terminal tothe first residue of SEQ ID NO: 2.

In some embodiments, when the composite peptide of SEQ ID NO: 2 is partof a longer peptide sequence, none of the amino acids joined througheach intra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position C terminal to the last residue of SEQ ID NO: 2. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Cterminal to the last residue of SEQ ID NO: 2. In some embodiments, 0, 1,2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position C terminal tothe last residue of SEQ ID NO: 2.

In some embodiments, when the composite peptide of SEQ ID NO: 2 is partof a longer peptide sequence, none of the amino acids joined througheach intra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position N terminal to the first residue of SEQ ID NO: 2 and at aposition C terminal to the last residue SEQ ID NO: 2. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Nterminal to the first residue of SEQ ID NO: 2 and at a single position Cterminal to the last residue of SEQ ID NO: 2. In some embodiments, 0, 1,2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position N terminal tothe first residue of SEQ ID NO: 2 and at a position C terminal to thelast residue of SEQ ID NO: 2.

In some embodiments of the composite peptide of SEQ ID NO: 2, none orone or more of the amino acids are joined through each intra-peptidehydrocarbon linker element positioned on the one or more additionalmoieties conjugated to one or more of the residues of SEQ ID NO: 2. Insome embodiments, 0 or 1 of the amino acids joined through eachintra-peptide hydrocarbon linker element may be positioned on the one ormore additional moieties conjugated to SEQ ID NO: 2. In someembodiments, 0, 1, 2, 3, 4 or 5 of the amino acids joined through eachintra-peptide hydrocarbon linker element may be positioned on the one ormore additional moieties conjugated to SEQ ID NO: 2.

In some embodiments of the composite peptide of SEQ ID NO: 2, 0, 1, or 2of the amino acids joined through each intra-peptide hydrocarbon linkerelement may include natural or non-natural amino acids in the (D) aswell as (L), or the (R) as well as (S), stereochemical configuration. Insome embodiments of the composite peptide of SEQ ID NO: 2, 0, 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 of the amino acids joined through eachintra-peptide hydrocarbon linker element comprise natural amino acids,non-natural amino acids, or a combination thereof. In some embodimentsof the composite peptide of SEQ ID NO: 2, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 of the amino acids joined through each intra-peptide hydrocarbonlinker element comprise the (D) stereochemical configuration, the (L)stereochemical configuration, or a combination thereof. In someembodiments of the composite peptide of SEQ ID NO: 2, 0, 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 of the amino acids joined through each intra-peptidehydrocarbon linker element comprise the (R) stereochemicalconfiguration, the (S) stereochemical configuration, or a combinationthereof.

In some embodiments of the composite peptide of SEQ ID NO: 2, 0, 1, or 2of the amino acids joined through each intra-peptide hydrocarbon linkerelement are connected through a substituted side chain alpha carbon ornon-substituted side chain alpha carbon. In some embodiments of thecomposite peptide of SEQ ID NO: 2, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ofthe amino acids joined through each intra-peptide hydrocarbon linkerelement are connected through a substituted side chain alpha carbon, anon-substituted side chain alpha carbon or a combination thereof.

Several embodiments relate to custom peptide derivatives of the γc-boxmotifs of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7, which are depicted inFIG. 1A. Other embodiments relate to custom derivative peptides whichare artificial composite peptides combining the amino acid sequence oftwo or more of the human IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7 γc-boxmotifs. Several embodiments relate to custom peptide derivatives of theγc-box motifs of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7 having apartial amino acid sequence that shows approximately 50%, 50-60%,60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to aminoacid sequences of the of the γc-box motifs of IL-15, IL-2, IL-21, IL-4,IL-9, or IL-7. Custom peptide derivatives of the of the γc-box motifs ofIL-15, IL-2, IL-21, IL-4, IL-9, or IL-7 further include any peptidewherein a partial amino acid sequence of that peptide derivativecomprises amino acids with similar physico-chemical properties to theamino acids of the γc-box motif sequences of IL-15, IL-2, IL-21, IL-4,IL-9, or IL-7.

Several embodiments relate to custom peptide derivatives that wouldinhibit the function of one, all, or selective members of theγc-cytokines. In some embodiments, the custom peptide derivativesselectively target individual γc-cytokine family members. For example, acustom peptide derivative can selectively inhibit the function of IL-2,IL-4, IL-7, IL-9, IL-15, or IL-21. In other embodiments, a custompeptide derivative can inhibit 2 or more γc-cytokine family members.

For example, the custom peptide derivatives of the present embodimentscan selectively inhibit the function of IL-2 in combination with one ormore of IL-4, IL-7, IL-9, IL-15, and IL-21; IL-4 in combination with oneor more of IL-7, IL-9, IL-15, and IL-21; IL-7 in combination with one ormore of IL-9, IL-15, and IL-21; IL-9 in combination with one or more ofIL-2, IL-4, IL-7, IL-15, and IL-21; IL-15 in combination with one ormore of IL-2, IL-4, IL-7, IL-9, and IL-21; or IL-21 in combination withone or more of IL-2, IL-4, IL-7, IL-9, and IL-15. In other embodiments,custom peptide derivatives can comprehensively target all γc-cytokinefamily members.

Not wishing to be bound by a particular theory, the custom peptidederivatives can inhibit the function of all or selective members of theγc-cytokines by diminishing the binding of γc-cytokines to theγc-subunit, for example, as a competitive inhibitor. Such custom peptidederivatives may be used in diverse applications, including as a clinicaldrug.

Several embodiments relate to custom peptide derivatives that wouldmodulate (including enhance or reduce) the function of one, two, or moreof selective members of the γc-cytokines. In some embodiments, thecustom peptide derivatives selectively target individual γc-cytokinefamily members. For example, a custom peptide derivative can selectivelyenhance or inhibit the function of IL-2, IL-4, IL-7, IL-9, IL-15, orIL-21. In other embodiments, a custom peptide derivative can enhance orinhibit two or more γc-cytokine family members. In certain embodiments,custom peptide derivatives may compriseP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), which canenhance or inhibit the activity of one, two or more of γc-cytokines. Incertain embodiments, custom peptide derivatives may compriseP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), which caninhibit the activity of at least IL-15 and IL-21.

In some embodiments, one or more of the custom peptide derivatives ofthe conserved γc-box motif disclosed herein can inhibit the activity ofone or more γc-cytokines. In some embodiments, one or more of the custompeptide derivatives of the conserved γc-box motif disclosed herein caninhibit the activity of one or more γc-cytokines by suppressing cellproliferation induced by the one or more γc-cytokines. In someembodiments, one or more of the custom peptide derivatives of theconserved γc-box motif disclosed herein can inhibit the activity of oneor more γc-cytokines by inhibiting phosphorylation of the intracellularcytokine signal transduction molecule mediated by the one or moreγc-cytokines. In some embodiments, one or more of the custom peptidederivatives of the conserved γc-box motif disclosed herein can inhibitthe activity of one or more γc-cytokines by suppressing cellproliferation induced by the one or more γc-cytokines and by inhibitingphosphorylation of the intracellular cytokine signal transductionmolecule mediated by the one or more γc-cytokines. In some embodiments,one or more of the custom peptide derivatives of the conserved γc-boxmotif disclosed herein can inhibit the activity of one or moreγc-cytokines by one or more other mechanisms.

In some embodiments, one or more of the peptide sequences disclosedherein suppress proliferation of one or more cell types induced by oneor more of the cytokines disclosed herein (e.g., IL-2, IL-4, IL-7, IL-9,IL-15, and IL-21). In some embodiments, one or more of the peptidesequences disclosed herein suppress proliferation of one or more celltypes induced by all of the cytokines disclosed herein. In someembodiments, one or more of the peptide sequences disclosed hereinsuppress proliferation of one or more cell types induced by some but notall of the cytokines disclosed herein. For example, the 21-mer SEQ IDNO: 3 suppressed IL-15 and IL-21 induced cellular proliferation, but notIL-2, IL-4, or IL-9 induced cellular proliferation. See FIG. 3C andEXAMPLE 2.

In some embodiments, one or more of the custom peptide derivatives ofthe conserved γc-box motif disclosed herein can inhibit the activity ofone or more γc-cytokines by inhibiting phosphorylation of one or moreintracellular cytokine signal transduction molecules mediated by the oneor more γc-cytokines disclosed herein (e.g., IL-2, IL-4, IL-7, IL-9,IL-15, and IL-21). In some embodiments, one or more of the custompeptide derivatives of the conserved γc-box motif disclosed herein caninhibit phosphorylation of one or more intracellular cytokine signaltransduction molecules mediated by all of the γc-cytokines disclosedherein. In some embodiments, one or more of the custom peptidederivatives of the conserved γc-box motif disclosed herein can inhibitphosphorylation of one or more intracellular cytokine signaltransduction molecules mediated by some but not all of the γc-cytokinesdisclosed herein. For example, SEQ ID NO: 3 inhibited IL-15 mediatedphosphorylation of the intracellular cytokine signal transductionmolecule, STAT-5, but not IL-2 mediated phosphorylation of STAT-5.

Also, for example, SEQ ID NO: 3 inhibited IL-21 mediated phosphorylationof the intracellular cytokine signal transduction molecule STAT-3. SeeFIG. 4B and EXAMPLE 5.

In some embodiments, custom peptide derivatives may include any peptidewhose partial amino acid sequence shows approximately 50%, 50-60%,60-70%, 70-80%, 80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to aminoacid sequence: P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).Custom peptide derivatives further include any peptide wherein a partialamino acid sequence of that peptide derivative comprises amino acidswith similar physico-chemical properties to the amino acids of sequence:P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).

In several embodiments, the amino acid residues of the custom derivativepeptides retain similar physico-chemical properties with the amino acidresidues of P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3),but exhibit different biological inhibition specificity to the 6γc-cytokine family members (i.e. IL-2, IL-4, IL-7, IL-9, IL-15, orIL-21) from that of the original peptide ofP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3). Peptidederivatives of the sequence of P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S(SEQ ID NO: 3) may be 19, 20, 21, 22, 23, 24, 25-30, 30-35, 35-40,40-45, 45-50, or more than 50 amino acids in length.

In some embodiments, the custom peptide derivatives may be conjugated tothe N-termini, C-termini and/or to the side residues of existingbiological proteins/peptides. In some embodiments, the composite peptideof SEQ ID NO: 3 may be conjugated to other moieties through theN-terminus, C-terminus or side chains of the composite peptide. Theother moieties may include proteins or peptides that stabilize thecomposite peptide, or other moieties, including without limitation,bovine serum albumin (BSA), albumin, Keyhole Limpet Hemocyanin (KLH), Fcregion of IgG, a biological protein that functions as scaffold, anantibody against a cell-specific antigen, a receptor, a ligand, a metalion and Poly Ethylene Glycol (PEG).

In some embodiments, any of the custom peptide derivatives disclosedherein can comprise one or more intra-peptide hydrocarbon linkerelements. In some embodiments, the composite peptide of SEQ ID NO: 3comprises one or more intra-peptide hydrocarbon linker elements. In someembodiments, the composite peptide of SEQ ID NO: 3 comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 4 residues apart on SEQ ID NO: 3. In someembodiments, the composite peptide of SEQ ID NO: 3 comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 7 residues apart on SEQ ID NO: 3. In someembodiments, the composite peptide of SEQ ID NO: 3 comprises one or moreintra-peptide hydrocarbon linker elements that connect two separateamino acids positioned 4 residues apart on SEQ ID NO: 3 and 7 residuesapart on SEQ ID NO: 3.

In some embodiments, when the composite peptide of SEQ ID NO: 3 is partof a longer peptide sequence, none of the amino acids joined througheach intra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position N terminal to the first residue of SEQ ID NO: 3. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Nterminal to the first residue of SEQ ID NO: 3. In some embodiments, 0,1, 2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position N terminal tothe first residue of SEQ ID NO: 3.

In some embodiments, when the composite peptide of SEQ ID NO: 3 is partof a longer peptide sequence, none of the amino acids joined througheach intra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position C terminal to the last residue of SEQ ID NO: 3. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Cterminal to the last residue of SEQ ID NO: 3. In some embodiments, 0, 1,2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position C terminal tothe last residue of SEQ ID NO: 3.

In some embodiments, when the composite peptide of SEQ ID NO: 3 is partof a longer peptide sequence, none of the amino acids joined througheach intra-peptide hydrocarbon or one or more of the amino acids joinedthrough each intra-peptide hydrocarbon linker element may be located ata position N terminal to the first residue of SEQ ID NO: 3 and at aposition C terminal to the last residue SEQ ID NO: 3. In someembodiments, 0 or 1 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a single position Nterminal to the first residue of SEQ ID NO: 3 and at a single position Cterminal to the last residue of SEQ ID NO: 3. In some embodiments, 0, 1,2, 3, 4 or 5 of the amino acids joined through each intra-peptidehydrocarbon linker element may be located at a position N terminal tothe first residue of SEQ ID NO: 3 and at a position C terminal to thelast residue of SEQ ID NO: 3.

In some embodiments of the composite peptide of SEQ ID NO: 3, none orone or more of the amino acids are joined through each intra-peptidehydrocarbon linker element positioned on the one or more additionalmoieties conjugated to one or more of the residues of SEQ ID NO: 3. Insome embodiments, 0 or 1 of the amino acids joined through eachintra-peptide hydrocarbon linker element may be positioned on the one ormore additional moieties conjugated to SEQ ID NO: 3. In someembodiments, 0, 1, 2, 3, 4 or 5 of the amino acids joined through eachintra-peptide hydrocarbon linker element may be positioned on the one ormore additional moieties conjugated to SEQ ID NO: 3.

In some embodiments of the composite peptide of SEQ ID NO: 3, 0, 1, or 2of the amino acids joined through each intra-peptide hydrocarbon linkerelement may include natural or non-natural amino acids in the (D) aswell as (L), or the (R) as well as (S), stereochemical configuration. Insome embodiments of the composite peptide of SEQ ID NO: 3, 0, 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 of the amino acids joined through eachintra-peptide hydrocarbon linker element comprise natural amino acids,non-natural amino acids, or a combination thereof. In some embodimentsof the composite peptide of SEQ ID NO: 3, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 of the amino acids joined through each intra-peptide hydrocarbonlinker element comprise the (D) stereochemical configuration, the (L)stereochemical configuration, or a combination thereof. In someembodiments of the composite peptide of SEQ ID NO: 3, 0, 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 of the amino acids joined through each intra-peptidehydrocarbon linker element comprise the (R) stereochemicalconfiguration, the (S) stereochemical configuration, or a combinationthereof.

In some embodiments of the composite peptide of SEQ ID NO: 3, 0, 1, or 2of the amino acids joined through each intra-peptide hydrocarbon linkerelement are connected through a substituted side chain alpha carbon ornon-substituted side chain alpha carbon. In some embodiments of thecomposite peptide of SEQ ID NO: 3, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ofthe amino acids joined through each intra-peptide hydrocarbon linkerelement are connected through a substituted side chain alpha carbon, anon-substituted side chain alpha carbon or a combination thereof.

Peptides of the present embodiments may also contain one or more rareamino acids (such as 4-hydroxyproline or hydroxylysine), organic acidsor amides and/or derivatives of common amino acids, such as amino acidshaving the C-terminal carboxylate esterified (e.g., benzyl, methyl orethyl ester) or amidated and/or having modifications of the N-terminalamino group (e.g., acetylation or alkoxycarbonylamino), with or withoutany of a wide variety of side chain modifications and/or substitutions.Side chain modifications, substitutions or a combination thereof thatmay be present in the custom peptide derivatives of the presentembodiments include, but are not limited to, α-methyl, α-alkenyl,alkylation, methylation, benzylation, t-butylation, tosylation,alkoxycarbonylamino, and the like.

Residues other than common amino acids that may be present include, butare not limited to, penicillamine, tetramethylene cysteine,pentamethylene cysteine, mercaptopropionic acid,pentamethylene-mercaptopropionic acid, 2-mercaptobenzene,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,aminoadipic acid, m-aminomethylbenzoic acid, and diaminopropionic acid.

Peptides of the present embodiments can be produced and obtained byvarious methods known to those skilled in the art. For example, thepeptide may be produced by genetic engineering, based on the nucleotidesequence coding for the peptide of the present embodiments, orchemically synthesized by means of peptide solid-phase synthesis and thelike, or produced and obtained in their combination. One skilled in theart of solid-phase peptide synthesis can readily incorporate natural ornon-natural amino acids in the (D) as well as (L), or the (R) as well as(S), stereochemical configuration. It will also be apparent to oneskilled in the art of solid-phase peptide synthesis to produce andobtain peptides containing one or more intra-peptide hydrocarbon linkerelements of the present embodiments utilizing a-substituted (such asα-alkenyl) natural or non-natural amino acids in one or more of (D),(L), (R) or (S), stereochemical configurations, or a combinationthereof. In some embodiments, an intra-peptide hydrocarbon linkerelement linking a-substituted amino acids (e.g., α-alkenyl amino acids)can be generated by catalyzing one or more ring-closing metathesis. Insome embodiments, one or more intra-peptide hydrocarbon linker elementscan be generated by catalyzing a ring-closing metathesis usingbenzylidenebis(tricyclohexyl-phosphine)-dichlororuthenium (Grubb'scatalyst) on the resin-bound peptide during peptide synthesis. In someembodiments, other ring-closing synthesis reactions and/or mechanismsduring one or more known peptide synthesis processes are alsocontemplated. In some embodiments, one or more inter-peptide hydrocarbonlinker elements can be generated by catalyzing a ring-closing metathesisusing benzylidenebis(tricyclohexyl-phosphine)-dichlororuthenium (Grubb'scatalyst) on the resin-bound peptide during peptide synthesis. In someembodiments, other ring-closing synthesis reactions and/or mechanismsduring one or more known peptide synthesis processes are alsocontemplated.

In some embodiments, at least two alpha-alkenyl substituted amino acidsin a composite peptide are linked by the at least one intra-peptidehydrocarbon linker element. In some embodiments, the at least twoalpha-alkenyl substituted amino acids linked by an intra-peptidehydrocarbon linker element are separated by n−2 amino acids, wherein nrepresents the number of amino acids encompassed by the intra-peptidelinkage.

In some embodiments, one or more intra-peptide hydrocarbon linkerelements are incorporated at amino acid positions that correlate with asingle α-helical turn in a secondary structure of the composite peptide.In some embodiments, when the composite peptide comprises one or morenon-contiguous single α-helical turns, the amino acid positions thatcorrelate with a single α-helical turn of the composite peptidecorrespond to amino acid positions i and i+4 of the composite peptide,where i is the first amino acid position of the single α-helical turnand i+4 is the last amino acid position of the single a-helical turn,and wherein amino acid positions i and i+4 comprise alpha-alkenylsubstituted amino acids, and where i and i+4 are positioned 4 residuesapart (4 spaced).

In some embodiments, one skilled in the art of solid-phase peptidesynthesis can readily synthesize composite peptides comprising more thanone intra-peptide hydrocarbon linker elements such that the compositepeptide comprises more than one single α-helical turn. In someembodiments, the more than one single α-helical turns arenon-contiguous, i.e., the more than one single α-helical turns do notshare a substituted amino acid. For example, in some embodiments, thecomposite peptide can comprise one or more intra-peptide hydrocarbonlinker elements of Formula 1, Formula 2, Formula 3, Formula 4, Formula5, Formula 6, Formula 7, and/or Formula 8 (See TABLE 1) that span morethan one non-contiguous single α-helical turns of the composite peptide.

Not wishing to be bound to any specific peptide containing one or moreintra-peptide hydrocarbon linker elements of the present embodiments, ageneric peptide example containing one intra-peptide hydrocarbon linkerelement connecting two separate amino acids positioned 4 residues apart,or one α-helical turn (position i and position i+4), can haveS-pentenylalanine (S5Ala) incorporated at each of the positions i andi+4 during solid-phase synthesis of the peptide before catalyzingring-closing metathesis using Grubb's catalyst while the peptide isstill resin-bound on the solid support. This will result in a peptidesequence containing the intra-peptide hydrocarbon linker elementdepicted below (SEQ ID NO: 78) positioned 4 residues apart:

In some embodiments, one or more intra-peptide hydrocarbon linkerelements are incorporated at amino acid positions that correlate with adouble α-helical turn in a secondary structure of the composite peptide.In some embodiments, when the composite peptide comprises one or morenon-contiguous double α-helical turns, the amino acid positions thatcorrelate with a double α-helical turn of the composite peptidecorrespond to amino acid positions i and i+7 of the composite peptide,where i is the first amino acid position of the double α-helical turnand i+7 is the last amino acid position of the double a-helical turn,and wherein amino acid positions i and i+7 comprise alpha-alkenylsubstituted amino acids, and where i and i+7 are positioned 7 residuesapart (7 spaced).

Not wishing to be bound to any specific peptide containing one or moreintra-peptide hydrocarbon linker elements of the present embodiments, ageneric peptide example containing one intra-peptide hydrocarbon linkerelement connecting two separate amino acids positioned 7 residues apart,or two α-helical turns (position i and position i+7), can haveR-octenylalanine (R8Ala) incorporated at position i andS-pentenylalanine (S5Ala) incorporated at position i+7 duringsolid-phase synthesis of the peptide before catalyzing ring-closingmetathesis using Grubb's catalyst while the peptide is still resin-boundon the solid support. This will result in a peptide sequence containingthe intra-peptide hydrocarbon linker elements depicted below (SEQ ID NO:79) positioned 7 residues apart:

It will be appreciated by one of ordinary skill in the art that thepositions of octenylalanine (R8Ala) and S-pentenylalanine (S5Ala) in SEQID NO: 79 can be switched such that S-pentenylalanine (S5Ala) isincorporated at position i and R-octenylalanine (R8Ala) is incorporatedat position i+7 (SEQ ID NO: 80).

Not wishing to be bound to any specific peptide containing one or moreintra-peptide hydrocarbon linker elements of the present embodiments, ageneric peptide example containing one intra-peptide hydrocarbon linkerelement connecting two separate amino acids positioned 7 residues apart,or two α-helical turns (position i and position i+7), can haveS-octenylalanine (S8Ala) incorporated at position i andR-pentenylalanine (R5Ala) incorporated at position i+7 duringsolid-phase synthesis of the peptide before catalyzing ring-closingmetathesis using Grubb's catalyst while the peptide is still resin-boundon the solid support. This will result in a peptide sequence containingthe intra-peptide hydrocarbon linker elements depicted below (SEQ ID NO:81) positioned 7 residues apart:

It will be appreciated by one of ordinary skill in the art that thepositions of S-octenylalanine (S8Ala) and R-pentenylalanine (R5Ala) inSEQ ID NO: 81 can be switched such that R-pentenylalanine (R5Ala) isincorporated at position i and S-octenylalanine (S8Ala) is incorporatedat position i+7 (SEQ ID NO: 82).

In some embodiments, one skilled in the art of solid-phase peptidesynthesis can readily synthesize composite peptides comprising more thanone intra-peptide hydrocarbon linker elements such that the compositepeptide comprises more than one double α-helical turn. In someembodiments, the more than one double α-helical turns arenon-contiguous, i.e., the more than one double α-helical turns do notshare a substituted amino acid. For example, in some embodiments, thecomposite peptide can comprise one or more intra-peptide hydrocarbonlinker elements of Formula 17, Formula 18, Formula 19, and/or Formula 20(See TABLE 1) that span more than one non-contiguous double α-helicalturns of the composite peptide.

One skilled in the art of solid-phase peptide synthesis can readilysynthesize peptides containing more than one intra-peptide hydrocarbonlinker element of the present embodiments by incorporating α-alkenylsubstituted amino acids at paired non-overlapping amino acid positionsin the peptide, with each α-alkenyl substituted amino acid in the pairpositioned a single α-helical turn apart (4 residues apart) or a doubleα-helical turn apart (7 residues apart) during solid-phase peptidesynthesis before catalyzing ring-closing metathesis using Grubb'scatalyst while the peptide is still resin-bound on the solid support. Insome embodiments, single peptides can comprise more than oneintra-peptide hydrocarbon linker element that span a single α-helicalturn (4 residues apart), can contain hydrocarbon linker elements thatspan a double α-helical turns (7 residues apart), or can contain acombination of both a single α-helical turn (4 residues apart) and adouble α-helical turn (7 residues apart) intra-peptide hydrocarbonlinker elements.

In some embodiments, when the composite peptide comprises two or morecontiguous single α-helical turns, the amino acid positions thatcorrelate with the first single α-helical turn of the composite peptidecorrespond to amino acid positions i and i+4 of the composite peptide,where i is the first amino acid position of the first single α-helicalturn and i+4 is the last amino acid position of the first singleα-helical turn, and the amino acid positions that correlate with thesecond single α-helical turn of the composite peptide correspond toamino acid positions i+4 and i+8 of the composite peptide, where i+4 isthe first amino acid position of the second single α-helical turn andi+8 is the last amino acid position of the second single α-helical turn,and wherein amino acid positions i, i+4 and i+8 comprise alpha-alkenylsubstituted amino acids.

In some embodiments, one skilled in the art of solid-phase peptidesynthesis can readily synthesize composite peptides comprising more thanone intra-peptide hydrocarbon linker elements such that the compositepeptide comprises more than one single α-helical turn. In someembodiments, the more than one single α-helical turns are contiguous,i.e., the more than one single α-helical turns share a substituted aminoacid. Thus, when two single α-helical turns are contiguous, the lastposition of a preceding single α-helical turn is the first positon of asubsequent contiguous single α-helical turn. In some embodiments, theshared a substituted amino acid is a di-substituted amino acid (SeeTABLE 1).

Thus, in some embodiments, the composite peptide can comprise one ormore intra-peptide hydrocarbon linker elements of Formula 9, Formula 10,Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, and/orFormula 16 (See TABLE 1) that span two contiguous single α-helical turnsof the composite peptide.

For example, in some embodiments, one skilled in the art of solid-phasepeptide synthesis can readily synthesize a peptide containing more thanone intra-peptide hydrocarbon linker element of the present embodimentsby incorporating a di-substituted a-alkenyl glycine amino acid aposition that is equidistant from one α-alkenyl substituted amino acidpositioned a single α-helical turn apart (4 residues apart) toward theN-terminus of the peptide, and another α-alkenyl substituted amino acidpositioned a single α-helical turn (4 residues apart) toward theC-terminus of the peptide during solid-phase peptide synthesis beforecatalyzing ring-closing metathesis using Grubb's catalyst while thepeptide is still resin-bound on the solid support.

In some embodiments, when the composite peptide comprises two or morecontiguous double α-helical turns, the amino acid positions thatcorrelate with the first double α-helical turn of the composite peptidecorrespond to amino acid positions i and i+7 of the composite peptide,where i is the first amino acid position of the first double α-helicalturn and i+7 is the last amino acid position of the first doubleα-helical turn, and the amino acid positions that correlate with thesecond double α-helical turn of the composite peptide correspond toamino acid positions i+7 and i+14 of the composite peptide, where i+7 isthe first amino acid position of the second double α-helical turn andi+14 is the last amino acid position of the second double α-helicalturn, and wherein amino acid positions i, i+7 and i+14 comprisealpha-alkenyl substituted amino acids.

In some embodiments, one skilled in the art of solid-phase peptidesynthesis can readily synthesize composite peptides comprising more thanone intra-peptide hydrocarbon linker elements such that the compositepeptide comprises more than one double α-helical turn. In someembodiments, the more than one double α-helical turns are contiguous,i.e., the more than one double α-helical turns share a substituted aminoacid. Thus, when two double α-helical turns are contiguous, the lastposition of a preceding double α-helical turn is the first positon of asubsequent contiguous double α-helical turn. In some embodiments, theshared a substituted amino acid is a di-substituted amino acid (SeeTABLE 1).

Thus, in some embodiments, the composite peptide can comprise one ormore intra-peptide hydrocarbon linker elements of Formula 21, Formula22, and/or Formula 23 (See TABLE 1) that span two contiguous doubleα-helical turns of the composite peptide. When two double α-helicalturns are contiguous, the last position of a preceding double a-helicalturn is the first positon of a subsequent contiguous double α-helicalturn.

For example, in some embodiments, one skilled in the art of solid-phasepeptide synthesis can readily synthesize a peptide containing more thanone intra-peptide hydrocarbon linker element of the present embodimentsby incorporating a di-substituted a-alkenyl glycine amino acid at aposition equidistant from one α-alkenyl substituted amino acidpositioned a double α-helical turn (7 residues apart) toward theN-terminus of the peptide, and another α-alkenyl substituted amino acidpositioned a double α-helical turn apart (7 residues apart) toward theC-terminus of the peptide during solid-phase peptide synthesis beforecatalyzing ring-closing metathesis using Grubb's catalyst while thepeptide is still resin-bound on the solid support.

Peptides containing one or more intra-peptide hydrocarbon linkerelements of the present embodiments can be produced through solid-phasepeptide synthesis utilizing commercially available Boc- orFmoc-protected α-alkenyl substituted natural or non-natural amino acidsin the (D) as well as (L), or the (R) as well as (S), stereochemicalconfiguration. The Fmoc-protected α-alkenyl substituted amino acids andthe resultant hydrocarbon linker element following ring-closingmetathesis that may be used in the synthesis of the custom peptidederivatives of the present embodiments include, but are not limited toTABLE 1:

TABLE 1 α-alkenyl Substituted Amino α-alkenyl Substituted Aminoα-alkenyl Substituted Acid Acid Amino Acid Peptide Position i PeptidePosition i + 4 Peptide Position i + 8 R-propenylalanine (CAS:R-pentenylalanine (CAS: N/A 288617-76-5; R3Ala), S- 288617-77-6; R5Ala),S- propenylalanine (CAS: pentenylalanine (CAS: 288617-71-0; S3Ala), D-288617-73-2; S5Ala), R- allylglycine (CAS: 170642-28- pentenylglycine(CAS: 1; D3Gly), or L-allylglycine 1093645-21-6; R5Gly), or 5- (CAS:146549-21-5; L3Gly) pentenylglycine (CAS: 856412-22-1; S5Gly)Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 1 R5Ala, S5Ala, R5Gly, or R3Ala, S3Ala, D3Gly, or N/A S5GlyL3Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 2 R-butenylalanine (CAS: R4Ala, S4Ala, R4Gly, or N/A1311933-82-0; R4Ala), S- S4Gly butenylalanine (CAS: 288617- 72-1;S4Ala), R- butenylglycine (CAS: 865352- 21-2; R4Gly), or S-butenylglycine (CAS: 851909- 08-5; S4Gly) Hydrocarbon Linker ElementFollowing Ring-Closing Metathesis

Formula 3 R5Ala, S5Ala, R5Gly, or R5Ala, S5Ala, R5Gly, or N/A S5GlyS5Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 4 R4Ala, S4Ala, R4Gly, or R-hexenylalanine (CAS: N/A S4Gly288617-78-7; R6Ala), S- hexenylalanine (CAS: 288617-74-3; S6Ala), R-hexenylglycine (CAS: 1208226-88-3; R6Gly), or S- hexenylglycine (CAS:1251904-51-4; S6Gly) Hydrocarbon Linker Element Following Ring-ClosingMetathesis

Formula 5 R6Ala, S6Ala, R6Gly, or R4Ala, S4Ala, R4Gly, or N/A S6GlyS4Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 6 R3Ala, S3Ala, D3Gly, or R-heptenylalanine (CAS: N/A L3Gly1311933-84-2; R7Ala), S- heptenylalanine (CAS: 1311933-83-1; S7Ala), R-heptenylglycine (CAS: 1262886-63-4; R7Gly), or S- heptenylglycine (CAS:1058705-57-9; S7Gly) Hydrocarbon Linker Element Following Ring-ClosingMetathesis

Formula 7 R7Ala, S7Ala, R7Gly, or R3Ala, S3Ala, D3Gly, or N/A S7GlyL3Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 8 R5Ala, S5Ala, R5Gly, or di-substituted bis- R5Ala, S5Ala,R5Gly, or S5Gly propenylglycine (CAS: S5Gly 1311992-97-8; bis3Gly)Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 9 R3Ala, S3Ala, D3Gly, or di-substituted bis- R3Ala, S3Ala,D3Gly, or L3Gly pentenylglycine (CAS: L3Gly 1068435-19-7; bis5Gly)Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 10 R4Ala, S4Ala, R4Gly, or di-substituted bis- R4Ala, S4Ala,R4Gly, or S4Gly butenylglycine (bis4Gly) S4Gly Hydrocarbon LinkerElement Following Ring-Closing Metathesis

Formula 11 R5Ala, S5Ala, R5Gly, or bis5Gly R5Ala, S5Ala, R5Gly, or S5GlyS5Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 12 R6Ala, S6Ala, R6Gly, or bis4Gly R6Ala, S6Ala, R6Gly, or S6GlyS6Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 13 R4Ala, S4Ala, R4Gly, or di-substituted bis- R4Ala, S4Ala,R4Gly, or S4Gly hexenylglycine (bis6Gly) S4Gly Hydrocarbon LinkerElement Following Ring-Closing Metathesis

Formula 14 R7Ala, S7Ala, R7Gly, or bis3Gly R7Ala, S7Ala, R7Gly, or S7GlyS7Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 15 R3Ala, S3Ala, D3Gly, or di-substituted bis- R3Ala, S3Ala,D3Gly, or L3Gly heptenylglycine (bis7Gly) L3Gly Hydrocarbon LinkerElement Following Ring-Closing Metathesis

Formula 16 Peptide Position i Peptide Position i + 7 Peptide Positioni + 14 R5Ala, S5Ala, R5Gly, or R-octenylalanine (CAS: N/A S5Gly945212-26-0; R8Ala), S- octenylalanine (CAS: 288617- 75-4; S8Ala), R-octenylglycine (CAS: 1191429-20-5; R8Gly), or S- octenylglycine (CAS:1262886-64-5; S8Gly) Hydrocarbon Linker Element Following Ring-ClosingMetathesis

Formula 17 R8Ala, S8Ala, R8Gly, or R5Ala, S5Ala, R5Gly, or N/A S8GlyS5Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 18 R6Ala, S6Ala, R6Gly, or R7Ala, S7Ala, R7Gly, or N/A S6GlyS7Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 19 R7Ala, S7Ala, R7Gly, or R6Ala, S6Ala, R6Gly, or N/A S7GlyS6Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 20 R8Ala, S8Ala, R8Gly, or bis5Gly R8Ala, S8Ala, R8Gly, or S8GlyS8Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 21 R7Ala, S7Ala, R7Gly, or bis6Gly R7Ala, S7Ala, R7Gly, or S7GlyS7Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 22 R6Ala, S6Ala, R6Gly, or bis7Gly R6Ala, S6Ala, R6Gly, or S6GlyS6Gly Hydrocarbon Linker Element Following Ring-Closing Metathesis

Formula 23

In some embodiments, an intra-peptide hydrocarbon linker can be furtherfunctionalized through one or more chemical reactions. In someembodiments, one or more carbon-carbon double bond(s) present in theintra-peptide hydrocarbon linker (e.g., Formula 1-Formula 23 in TABLE 1)can be utilized for organic chemical reactions to add one or moreadditional chemical functionalities. For example, alkene reactions maybe utilized for custom peptide derivatives that contain one or moreintra-peptide hydrocarbon linker elements of the present embodiments.Non-limiting examples of alkene reactions include hydroboration,oxymercuration, hydration, chlorination, bromination, addition of HF,HBr, HCl or HI, dihydroxylation, epoxidation, hydrogenation, andcyclopropanation. In some embodiments, one or more additional chemicalfunctionalities of the intra-peptide hydrocarbon linker elements can beachieved subsequent to the alkene reaction. Non-limiting examplesinclude covalent addition of one or more chemical group substituents,such as nucleophilic reactions with epoxide and hydroxyl groups, and thelike. In some embodiments, alkene reactions may be utilized to attachbiotin, radioisotopes, therapeutic agents (non-limiting examples includerapamycin, vinblastine, taxol, etc.), non-protein fluorescent chemicalgroups (non-limiting examples include FITC, hydrazide, rhodamine,maleimide, etc.), and protein fluorescent groups (non-limiting examplesinclude GFP, YFP, mCherry, etc.) to one or more inter- and/orintra-peptide hydrocarbon linker elements of the present embodiments.

In some embodiments, custom peptide derivatives comprising anintra-peptide hydrocarbon linker element spanning a single α-helicalturn (4 spaced) are provided. In some embodiments, custom peptidederivatives comprising an intra-peptide hydrocarbon linker elementspanning a double α-helical turn (7 spaced) are provided. In someembodiments, custom peptide derivatives comprising one or moreintra-peptide hydrocarbon linker elements synthesized utilizingα-alkenyl substituted amino acids by ring-closing can span any number ofamino acids. In some embodiments, custom peptide derivatives comprisingone or more intra-peptide hydrocarbon linker elements synthesizedutilizing a-alkenyl substituted amino acids by ring-closing span 2 toabout 200 amino acids. In some embodiments, custom peptide derivativescomprising one or more intra-peptide hydrocarbon linker elementssynthesized utilizing α-alkenyl substituted amino acids by ring-closingspan 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195 or 200 amino acids, or a numberwithin a range defined by any two of the aforementioned values.

Non-limiting examples of composite peptides comprising one or moreintra-peptide hydrocarbon linker elements are provided in TABLE 2. Theexamples in TABLE 2 are not limiting with respect to any specificα-alkenyl substituted amino acid useful for the synthesis of singleα-helical turn (4 spaced) and/or double α-helical turn (7 spaced)intra-peptide hydrocarbon linker elements of the present embodimentsand/or to any specific amino acid stereochemical configuration (e.g.,(D) stereochemical configuration denoted with “d” in TABLE 2) in thecustom peptide derivatives of the present embodiments.

TABLE 2 SEQ ID NO:{S5Ala}-P-K-E-{S5Ala}-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S 12{S5Ala}-K-E-F-{S5Ala}-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S 13P-{S5Ala}-E-F-L-{S5Ala}-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S 14P-K-{S5Ala}-F-L-E-{S5Ala}-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S 15P-K-E-{S5Ala}-L-E-R-{S5Ala}-V-H-L-V-Q-M-F-I-H-Q-S-L-S 16P-K-E-F-{S5Ala}-E-R-F-{S5Ala}-H-L-V-Q-M-F-I-H-Q-S-L-S 17P-K-E-F-L-{S5Ala}-R-F-V-{S5Ala}-L-V-Q-M-F-I-H-Q-S-L-S 18P-K-E-F-L-E-{S5Ala}-F-V-H-{S5Ala}-V-Q-M-F-I-H-Q-S-L-S 19P-K-E-F-L-E-R-{S5Ala}-V-H-L-{S5Ala}-Q-M-F-I-H-Q-S-L-S 20P-K-E-F-L-E-R-F-{S5Ala}-H-L-V-{S5Ala}-M-F-I-H-Q-S-L-S 21P-K-E-F-L-E-R-F-V-{S5Ala}-L-V-Q-{S5Ala}-F-I-H-Q-S-L-S 22P-K-E-F-L-E-R-F-V-H-{S5Ala}-V-Q-M-{S5Ala}-I-H-Q-S-L-S 23P-K-E-F-L-E-R-F-V-H-L-{S5Ala}-Q-M-F-{S5Ala}-H-Q-S-L-S 24P-K-E-F-L-E-R-F-V-H-L-V-{S5Ala}-M-F-I-{S5Ala}-Q-S-L-S 25P-K-E-F-L-E-R-F-V-H-L-V-Q-{S5Ala}-F-I-H-{S5Ala}-S-L-S 26P-K-E-F-L-E-R-F-V-H-L-V-Q-M-{S5Ala}-I-H-Q-{S5Ala}-L-S 27P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-{S5Ala}-H-Q-S-{S5Ala}-S 28P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-{S5Ala}-Q-S-L-{S5Ala} 29P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-{S5Ala}-S-L-S-{S5Ala} 30{R8Ala}-P-K-E-F-L-E-{S5Ala}-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S 31{R8Ala}-K-E-F-L-E-R-{S5Ala}-V-H-L-V-Q-M-F-I-H-Q-S-L-S 32P-{R8Ala}-E-F-L-E-R-F-{S5Ala}-H-L-V-Q-M-F-I-H-Q-S-L-S 33P-K-{R8Ala}-F-L-E-R-F-V-{S5Ala}-L-V-Q-M-F-I-H-Q-S-L-S 34P-K-E-{R8Ala}-L-E-R-F-V-H-{S5Ala}-V-Q-M-F-I-H-Q-S-L-S 35P-K-E-F-{R8Ala}-E-R-F-V-H-L-{S5Ala}-Q-M-F-I-H-Q-S-L-S 36P-K-E-F-L-{R8Ala}-R-F-V-H-L-V-{S5Ala}-M-F-I-H-Q-S-L-S 37P-K-E-F-L-E-{R8Ala}-F-V-H-L-V-Q-{S5Ala}-F-I-H-Q-S-L-S 38P-K-E-F-L-E-R-{R8Ala}-V-H-L-V-Q-M-{S5Ala}-I-H-Q-S-L-S 39P-K-E-F-L-E-R-F-{R8Ala}-H-L-V-Q-M-F-{S5Ala}-H-Q-S-L-S 40P-K-E-F-L-E-R-F-V-{R8Ala}-L-V-Q-M-F-I-{S5Ala}-Q-S-L-S 41P-K-E-F-L-E-R-F-V-H-{R8Ala}-V-Q-M-F-I-H-{S5Ala}-S-L-S 42P-K-E-F-L-E-R-F-V-H-L-{R8Ala}-Q-M-F-I-H-Q-{S5Ala}-L-S 43P-K-E-F-L-E-R-F-V-H-L-V-{R8Ala}-M-F-I-H-Q-S-{S5Ala}-S 44P-K-E-F-L-E-R-F-V-H-L-V-Q-{R8Ala}-F-I-H-Q-S-L-{S5Ala} 45P-K-E-F-L-E-R-F-V-H-L-V-Q-M-{R8Ala}-I-H-Q-S-L-S-{S5Ala} 46{dP}-{dK}-{dE}-{dF}-{dL}-E-R-{R8Ala}-V-H-L-V-Q-F-{S5Ala}-I-{dH}- 47{dQ}-{dS}-{dL}-{dS}{dP}-{dK}-{dE}-{dF}-{dL}-E-R-{R8Ala}-V-H-L-V-Q-F-{S5Ala}-I-{dH}- 48{dQ}-{dS}-{dL}-S{S5Ala₁}*-P-K-E-{S5Ala₁}-L-E-R-{R8Ala₂}-V-H-L-V-Q-F-{S5Ala₂}-I- 49{dH}-{dQ}-{dS}-{dL}-{dS}{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{R8Ala₂}-V-H-L-V-Q-F-{S5Ala₂}-I- 50{dH}-{dQ}-{dS}-{dL}-S{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{R8Ala₂}-V-H-L-V-Q-F-{S5Ala₂}- 51{S5Ala₃}-H-Q-S-{S5Ala₃}-{dS}{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{R8Ala₂}-V-H-L-V-Q-F-{S5Ala₂}- 52{S5Ala₃}-H-Q-S-{S5Ala₃}-S{dP}-{dK}-{dE}-{dF}-{dL}-E-R-{R8Ala₁}-V-H-L-V-Q-F-{S5Ala₁}- 53{S5Ala₂}-H-Q-S-{S5Ala₂}-{dS}{dP}-{dK}-{dE}-{dF}-{dL}-E-R-{R8Ala₁}-V-H-L-V-Q-F-{S5Ala₁}- 54{S5Ala₂}-H-Q-S-}S5Ala₂}-S{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{S5Ala₂}-V-H-L-{S5Ala₂}-Q-F-F-I- 55{dH}-{dQ}-{dS}-{dL}-{dS}{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{S5Ala₂}-V-H-L-{S5Ala₂}-Q-F-F-I- 56{dH}-{dQ}-{dS}-{dL}-S{dR}-{dR}-{dR}-{dR}-{dP}-{dK}-{dE}-{dF}-{S5Ala₁}-E-R-F-{R5Ala₁}- 57{dH}-{S5Ala₂}-V-Q-L-{S5Ala₂}-I-{dH}-{dQ}-{dS}-{dL}-{dS}{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{S5Ala₂}-V-H-L-{S5Ala₂}-Q-F-F- 58{S5Ala₃}-H-Q-S-{S5Ala₃}-{dS}{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{S5Ala₂}-V-H-L-{S5Ala₂}-Q-F-F- 59{S5Ala₃}-H-Q-S-{S5Ala₃}-S{dP}-{dK}-{dE}-{dF}-{dL}-E-R-{R8Ala₁}-V-H-L-V-Q-M-{S5Ala₁}-I- 60{dH}-{dQ}-{dS}-{dL}-S{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{R8Ala₂}-V-H-L-V-Q-M-{S5Ala₂}-I- 61{dH}-{dQ}-{dS}-{dL}-S{S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-{S5Ala₂}-V-H-L-{S5Ala₂}-Q-M-F- 62{S5Ala₃}-H-Q-S-{S5Ala₃}-S{S5Ala}-I-K-E-{S5Ala}-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S 63I-K-E-F-L-Q-R-{S5Ala}-I-H-I-{S5Ala}-Q-S-I-I-N-T-S 64I-K-E-F-L-Q-R-{R8Ala}-I-H-I-V-Q-S-{S5Ala}-I-N-T-S 65I-K-E-F-L-Q-R-F-I-H-I-{S5Ala}-Q-S-I-{S5Ala}-N-T-S 66I-K-E-F-L-Q-R-F-I-H-I-{R8Ala}-Q-S-I-I-N-T-{S5Ala} 67{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{S5Ala₂}-I-H-I-{S5Ala₂}-Q-S-I-I-N-T-S 68{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{R8Ala₂}-I-H-I-V-Q-S-{S5Ala₂}-I-N-T-S 69{S5Ala₁}-I-K-E-}S5Ala₁}-L-Q-R-F-I-H-I-{S5Ala₂}-Q-S-I-{S5Ala₂}-N-T-S 70{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-F-I-H-I-{R8Ala₂}-Q-S-I-I-N-T-{S5Ala₂} 71{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{S5Ala₂}-I-H-I-{S5Ala₂}-Q-S-I-I-{dN}- 72{dT}-{dS}{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{R8Ala₂}-I-H-I-V-Q-S-{S5Ala₂}-I-{dN}- 73{dT}-{dS}{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-F-I-H-I-{S5Ala₂}-Q-S-I-{S5Ala₂}-{dN}- 74{dT}-{dS}{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{S5Ala₂}-I-H-I-{bis5Gly_(2,3)}-Q-S-I- 75{S5Ala₃}-N-T-S{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{S5Ala₂}-I-H-I-{bis5Gly_(2,3)}-Q-S-I-I-N-76 T-{R8Ala₃}{S5Ala₁}-I-K-E-{S5Ala₁}-L-Q-R-{S5Ala₂}-I-H-I-{bis5Gly_(2,3)}-Q-S-I- 77{S5Ala₃}-{dN}-{dT}-{dS} {S5Ala₁}-P-K-E-{S5Ala₁}-L-E-R-F-V-H-L-V-Q-K- 83{S5Ala₂}-I-H-Q-{S5Ala₂}-L-S *Subscript denotes corresponding pairs ofhydrocarbon-linked α-alkenyl substituted amino acids

One skilled in the art can synthesize the custom peptide derivativeswith or without one or more hydrocarbon linker elements based on thepresent disclosure of the conserved γc-box motif and knowledge of thebiochemical properties of amino acids as described in FIG. 2. Someembodiments also relate to polynucleotides comprising nucleotidesequences encoding the peptides of the present invention. “Nucleotidesequence,” “polynucleotide,” or “nucleic acid” can be usedinterchangeably, and are understood to mean either double-stranded DNA,a single-stranded DNA or products of transcription of the said DNAs(e.g., RNA molecules). Polynucleotides can be administered to cells orsubjects and expressed by the cells or subjects, rather thanadministering the peptides themselves. Several embodiments also relateto genetic constructs comprising a polynucleotide sequence encoding thepeptides of the present invention. Genetic constructs can also containadditional regulatory elements such as promoters and enhancers and,optionally, selectable markers.

Methods of Treating γc-Cytokine Mediated Diseases

Several embodiments relate to the use of γc-antagonist peptides in thetreatment of γc-cytokine mediated diseases. Use of custom peptidederivative according to the present embodiments allows for flexibilityin the design of the therapeutic agent (custom design of the peptide)and enables more comprehensive outcomes, which would not be accomplishedby conventional strategies employing anti-cytokine or anti-cytokinereceptor antibodies.

Described herein is a novel method of blocking the action of γc-familycytokines. Such manipulations can yield effective methods of clinicalinterventions in treating diseases related to the dysregulation ordysfunction of γc-cytokines. Examples of disease that may be treated bydisrupting the interaction between the γc-cytokine and the γc-subunitinclude autoimmune diseases such as systemic lupus erythematosis,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease (CD),Hashimoto's or auto-immune thyroiditis; collagen diseases includingrheumatoid arthritis, inflammatory bowel disease, diabetes mellitus(e.g., type 1 diabetes mellitus), autoimmune diseases of the skin suchas psoriasis; degenerative neuronal diseases such as multiple sclerosis,uvietis or inflammation of the eye and sympathetic ophthalmia,graft-versus-host disease (GvHD), myasthenia gravis, inflammatory boweldiseases (IBD, including ulcerative colitis and Crohn's disease),Systemic Lupus Erythematosus, and alopecia areata.

In some embodiments, the γc-antagonist peptides described herein may beused in the treatment of Human T-cell Lymphotropic type I and II (HTLV-Iand HTLV-II)-associated diseases including Adult T-cell Leukemia (ATL),HTLV-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), andother non-neoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy andmyositis. In some embodiments, the γc-antagonist peptides describedherein may be used in the treatment of other viral diseases such asinfluenza, AIDS, HBV and Herpes or parasitic diseases.

In several embodiments, the γc-antagonist peptides may be administeredbefore, during, and or after transplantation of various organs as animmunosuppressant agent.

In some embodiments, the γc-antagonist peptides described herein may beused in the treatment of immune-mediated diseases such as asthma andother inflammatory respiratory diseases, such as, but not limited tosinusitis, hay fever, bronchitis, chronic obstructive pulmonary disease(COPD), allergic rhinitis, acute and chronic otitis, lung fibrosis. Insome embodiments, γc-antagonist peptides may be administered to treat orprevent allergic reactions due to exposure to allergens, chemical agentsor other common causes of acute respiratory disease. In someembodiments, γc-antagonist peptides may be administered to treat orprevent inflammatory responses caused by viruses, bacteria, chemicalreagents, and biochemical reagents.

In several embodiments, the γc-antagonist peptides may be administeredto treat some types of malignancies such as LGL-leukemia,Intraepithelial lymphoma and leukemia in Refractory Celiac Disease, NKleukemia/lymphoma and NK-T leukemia/lymphoma

In some embodiments, custom peptide derivatives according to theembodiments described herein can be used for cosmetic purposes, such asthe treatment of acne, hair loss, sunburn and nail maintenance, includedto ointment as anti-aging component because of the anti-inflammatorynature of them.

Several embodiments relate to therapeutic antagonist peptides that wouldinhibit the function of all or selective members of the γc-cytokines. Insome embodiments, therapeutic antagonist peptides selectively inhibitindividual γc-cytokine family members (custom peptides). In otherembodiments, therapeutic antagonist peptides can comprehensively inhibitall γc-cytokine family members (Simul-Block). In some embodiments,therapeutic antagonist peptides selectively inhibit subsets of theγc-cytokines. Not wishing to be bound by a particular theory, thepeptide antagonists can inhibit the function of all or selective membersof the γc-cytokines by diminishing the binding of γc-cytokines to theγc-subunit, for example, as a competitive inhibitor.

Several members of the γc-cytokine family, IL-2, IL-7, and IL-15, butnot IL-4 have been implicated as being involved in graft versus hostdisease (GvHD) in an experimental mouse model. (Miyagawa et al., 2008 J.Immunol. 181:1109-19.) One embodiment relates to the use of therapeuticantagonist peptides that selectively inhibit IL-2, IL-7, and IL-15activity for the treatment of GvHD in humans, allowing survival of thegrafted tissues or bone marrow cells. Other embodiments relate to theuse of therapeutic antagonist peptides that selectively inhibit acombination of IL-2 and IL-7, IL-2 and IL-15, or IL-7 and IL-15 to treatGvHD. Other embodiments relate to the use of a combination oftherapeutic antagonist peptides that selectively inhibit IL-2, IL-7, orIL-15.

Some embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-2 function for the treatment of autoimmunedisorders where T-regs have been implicated as playing a role. In someembodiments, peptide-mediated inhibition of T-regs can enhance thenatural anti-cancer immunity in humans, providing a novel means ofanti-cancer therapy.

Several embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-4 to treat asthma.

Some embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-7 either alone or in combination withtherapeutic antagonist peptides that selectively inhibit the γc-cytokinefamily member, IL-15, as a therapeutic agent for LGL leukemia. In someembodiments therapeutic antagonist peptides that selectively inhibitboth IL-7 and IL-15 activity can be used to treat LGL leukemia. Severalembodiments relate to the use of BNZ-γ to treat LGL leukemia. In someembodiments, specific γc-antagonist peptides that selectively inhibitIL-15 alone or specific γc-antagonist peptides that selectively inhibitIL-15 and IL-7 are used as a therapeutic agent for CD4/CD8 Tlymphocyte-associated leukemia including that caused by the HTLV-I.

Several embodiments relate to the use of γc-antagonist peptides thatselectively inhibit the activity of IL-9, either alone or in combinationwith the other γc-cytokine family members, as a therapeutic agent forhuman diseases that involve the abnormal development of Th17 cells.

Several embodiments relate to the use of therapeutic antagonist peptidesthat selectively inhibit IL-15 activity as a therapeutic agent fortreating CD. One publication suggested that IL-21, in addition to IL-15,may play a role in CD pathogenesis. (See Bodd et al., 2010, MucosalImmunol. 3:594-601.) Furthermore, a recent study also identifiedsynergistic effects of IL-2, IL-15 and IL-21 contribute greatly to thepathogenesis of refractory CD (Kooy-Winkelaar, et al., 2017 Proc NatlAcad Sci USA 114: E980-9.). This suggests that optimum treatment of CDby conventional anti-cytokine or cytokine-receptor antibodies wouldbenefit from a combination of at least two antibodies recognizing one ormore components that belong to the IL-2 system, IL-15 system, IL-21system, or a combination thereof. In some embodiments, custom derivativeantagonist peptides that selectively inhibit IL-2, IL-15, IL-21, acombination of IL-2 and IL-15, a combination of IL-2 and IL-21, and/or acombination of IL-15 and IL-21 activities are used as a therapeuticagent for treating CD. In some embodiments, the effect of customderivative antagonist peptides that selectively inhibit a combination ofIL-2 and IL-15, a combination of IL-2 and IL-21, and/or a combination ofIL-15 and IL-21 can be additive or synergistic.

An additive effect is observed when the effect of a combination is equalto the sum of the effects of the individuals in the combination (e.g.,the effect of a combination of two or more peptides is equal to the sumof the effects of peptides individually). A synergistic effect isobserved when the effect of a combination is greater than the sum of theeffects of the individuals in the combination (e.g., the effect of acombination of two or more peptides is greater than the sum of theeffects of peptides individually). A synergistic effect is greater thanan additive effect. Additive effect, synergistic effect, or both canoccur in human patients, non-human patients, non-patient humanvolunteers, in vivo models, ex vivo models, in vitro models, etc.

In some embodiments, two or more peptides disclosed herein can be usedin combination. In some embodiments, two or more peptides disclosedherein when used in combination yield an additive effect. In someembodiments, two or more peptides disclosed herein when used incombination yield a synergistic effect. Synergistic effect can rangefrom about >1 to about 100-fold. In some embodiments, the synergisticeffect is about 2 to about 20-fold. In some embodiments, the synergisticeffect is about 20 to about 100 fold. In some embodiments, thesynergistic effect is from >1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, or 100-fold, or within a range defined by any two ofthe aforementioned values.

In addition to having therapeutic applications, γc-antagonist peptideshave applications in consumer products as well. Several embodimentsrelate to the use of γc-antagonist peptides in skin care products suchas anti-aging, anti-inflammatory, anti-acne, and other relatedapplications. Some embodiments relate to the use of γc-antagonistpeptides in hair products as anti-hair loss ingredient to treat hairloss caused by autoimmune disorders.

Another embodiment relates to the development of chemical compounds(non-peptide, non-protein) that have a spatial structure which resemblesthe 19-mer amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1), or the 21-mer amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), and can fitinto the pocket of the γc-subunit to structurally hinder the access of aγc-cytokine to the γc-subunit for binding. Some embodiments relate tothe use of structurally similar chemical compounds as inhibitors ofγc-cytokine activity. Such molecular mimicry strategy to further refinethe development of synthetic compounds resembling in structure toexisting biological peptide/proteins is described in Orzaez et al., 2009Chem. Med. Chem. 4:146-160. Another embodiment relates to administrationof chemical compounds (non-peptide, non-protein) that have a resembling3D structure as the 19-mer amino acids sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), or the 21-meramino acid sequence P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ IDNO: 3), to treat γc-cytokine-mediated diseases.

Several embodiments relate to the administration of a peptide of aminoacid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) totreat γc-cytokine-mediated diseases. Another embodiment relates to theadministration of peptide derivatives of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), wherein the aminoacid sequence of the derivative peptide has similar physico-chemicalproperties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), but has distinctbiological activity, to treat γc-cytokine-mediated diseases. Anotherembodiment relates to administration of a peptide of amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) conjugated to theN- and C-termini or to the side residues of existing biologicalproteins/peptides into patients to treat γc-cytokine-mediated diseases.Another embodiment relates to administration of a peptide of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1) thatcomprises one or more intra-peptide hydrocarbon linker elements to treatγc-cytokine-mediated diseases.

Several embodiments relate to the administration of a peptide of aminoacid sequence P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3)to treat γc-cytokine-mediated diseases. Another embodiment relates tothe administration of peptide derivatives of amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S(SEQ ID NO: 3), wherein theamino acid sequence of the derivative peptide has similarphysico-chemical properties as a peptide of the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), but hasdistinct biological activity, to treat γc-cytokine-mediated diseases.Another embodiment relates to administration of a peptide of amino acidsequence P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3)conjugated to the N- and C-termini or to the side residues of existingbiological proteins/peptides into patients to treat γc-cytokine-mediateddiseases. Another embodiment relates to administration of a peptide ofamino acid sequence P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ IDNO: 3) that comprises one or more intra-peptide hydrocarbon linkerelements to treat γc-cytokine-mediated diseases.

Several embodiments relate to administration of polyclonal andmonoclonal antibodies raised against a peptide comprising of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), or raisedagainst a peptide comprising of amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), into patientsas an immunogen to treat γc-cytokine-mediated diseases. Anotherembodiment relates to administration of polyclonal and monoclonalantibodies that were raised against derivative peptides of amino acidsequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S(SEQ ID NO: 1), or raisedagainst derivative peptides of amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), wherein theamino acid sequence of the derivative peptide has similarphysico-chemical properties as a peptide of the amino acid sequenceI-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1), or has similarphysico-chemical properties as a peptide of the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), but hasdistinct biological activity, into patients as an immunogen to treatγc-cytokine-mediated diseases.

Administration of γc-Antagonist Peptides

The present embodiments also encompass the use of γc-antagonist peptidesfor the manufacture of a medicament for the treatment of a disease. Thepresent embodiments also encompass a pharmaceutical composition thatincludes γc-antagonist peptides in combination with a pharmaceuticallyacceptable carrier. The pharmaceutical composition can include apharmaceutically acceptable carrier and a non-toxic therapeuticallyeffective amount of γc-antagonist peptides, or other compositions of thepresent embodiments.

The present embodiments provide methods of using pharmaceuticalcompositions comprising an effective amount of antagonists forγc-cytokines in a suitable diluent or carrier. A γc-antagonist of thepresent embodiments can be formulated according to known methods used toprepare pharmaceutically useful compositions. A γc-antagonist can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically-suitable diluents (e.g.,phosphate, acetate, Tris-HCl), preservatives (e.g., thimerosal, benzylalcohol, parabens), emulsifying compounds, solubilizers, adjuvants,and/or carriers such as bovine serum albumin.

In some embodiments, one or more compositions and kits comprising one ormore of the composite peptides or derivatives thereof disclosed hereinare contemplated. In some embodiments, one or more compositions and kitsare used for preventing and/or treating one or more diseases. In someembodiments, one or more compositions and kits are used for preventingand/or treating a γc cytokine-mediated disease. In some embodiments, oneor more compositions and kits are used for preventing and/or treating anHTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP)associated disease. In some embodiments, one or more compositions andkits are used for preventing and/or treating an inflammatory respiratorydisease. In some embodiments, one or more compositions and kits are usedfor preventing and/or treating a cosmetic condition.

Some embodiments, the one or more compositions and kits comprising oneor more of the composite peptides are administered to a subject in needthereof via any of the routes of administration provided herein. In someembodiments, the one or more compositions and kits comprises one or moreof the composite peptides or derivatives thereof at a therapeuticallyeffective amount to modulate the activity of one or more γc-cytokinesselected from the group consisting of IL 2, IL 4, IL 7, IL 9, IL 15, andIL 21. In some embodiments, the one or more compositions and kitscomprises one or more of the composite peptides or derivatives thereofat a therapeutically effective amount to prevent and/or treat one ormore diseases. In some embodiments, the one or more compositions andkits comprising one or more of the composite peptides additionallycomprise one or more pharmaceutically acceptable carriers, diluents,excipients or combinations thereof.

In some embodiments, one or more composite peptides in the one or morecompositions and kits are formulated as suitable for administration to asubject for preventing and/or treating one or more diseases. In someembodiments, one or more composite peptides in the one or morecompositions and kits are formulated as suitable for administration to asubject for preventing and/or treating a γc cytokine-mediated disease.In some embodiments, one or more composite peptides in the one or morecompositions and kits are formulated as suitable for administration to asubject for preventing and/or treating an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease. In someembodiments, one or more composite peptides in the one or morecompositions and kits are formulated as suitable for administration to asubject for preventing and/or treating an inflammatory respiratorydisease. In some embodiments, one or more composite peptides in the oneor more compositions and kits are formulated as suitable foradministration to a subject for preventing and/or treating a cosmeticcondition.

In some embodiments, one or more composite peptides selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in theone or more compositions and kits are formulated as suitable foradministration to a subject for preventing and/or treating one or morediseases. In some embodiments, one or more composite peptides selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3 in the one or more compositions and kits are formulated as suitablefor administration to a subject for preventing and/or treating a γccytokine-mediated disease. In some embodiments, one or more compositepeptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3 in the one or more compositions and kits areformulated as suitable for administration to a subject for preventingand/or treating an HTLV-1-associated myelopathy (HAM)/tropical spasticparaparesis (TSP) associated disease. In some embodiments, one or morecomposite peptides selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, and SEQ ID NO: 3 in the one or more compositions and kitsare formulated as suitable for administration to a subject forpreventing and/or treating an inflammatory respiratory disease. In someembodiments, one or more composite peptides selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the one ormore compositions and kits are formulated as suitable for administrationto a subject for preventing and/or treating a cosmetic condition.

In some embodiments, one or more derivatives of the one or morecomposite peptides selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, and SEQ ID NO: 3 in the one or more compositions and kitsare formulated as suitable for administration to a subject forpreventing and/or treating one or more diseases. In some embodiments,one or more derivatives of the one or more composite peptides selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:3 in the one or more compositions and kits are formulated as suitablefor administration to a subject for preventing and/or treating a γccytokine-mediated disease. In some embodiments, one or more derivativesof the one or more composite peptides selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the one or morecompositions and kits are formulated as suitable for administration to asubject for preventing and/or treating an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease. In someembodiments, one or more derivatives of the one or more compositepeptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3 in the one or more compositions and kits areformulated as suitable for administration to a subject for preventingand/or treating an inflammatory respiratory disease. In someembodiments, one or more derivatives of the one or more compositepeptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3 in the one or more compositions and kits areformulated as suitable for administration to a subject for preventingand/or treating a cosmetic condition. In some embodiments, the one ormore derivatives of the one or more composite peptides comprise aminoacid sequences that shares about 50% to about 99% identity with the oneor more composite peptides. In some embodiments, the one or morederivatives of the one or more composite peptides comprise amino acidsequences that shares 50%, 50-60%, 60-70%, 70-80%, 80%, 90%, 95%, 97%,98%, 99% or 99.8% identity with the one or more composite peptides, orwithin a range defined by any two of the aforementioned values.

In some embodiments, one or more γc-cytokine-mediated disease isselected from the group consisting of CD4-leukemia, CD8-leukemia,LGL-leukemia, systemic lupus erythematosis, Sjögren's syndrome,Wegener's granulomatosis, Celiac disease, Hashimoto's thyroiditis,rheumatoid arthritis, diabetes mellitus, psoriasis, multiple sclerosis,uvietis, inflammation of the eye, and graft-versus-host disease (GvHD).In some embodiments, one or more HAM/TSP associated disease is selectedfrom the group consisting of Adult T-cell Leukemia (ATL),HTLV-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), andother non-neeoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy, andmyositis. In some embodiments, one or more inflammatory respiratorydisease is selected from the group consisting of asthma, sinusitis, hayfever, bronchitis, chronic obstructive pulmonary disease (COPD),allergic rhinitis, acute and chronic otitis, and lung fibrosis). In someembodiments, one or more cosmetic disease is selected from the groupconsisting of acne, hair loss, sunburn, nail maintenance, and appearanceof aging.

Suitable carriers and their formulations are described in Remington'sPharmaceutical Sciences, 16^(th) ed. 1980 Mack Publishing CO.Additionally, such compositions can contain a γc-antagonist complexedwith polyethylene glycol (PEG), metal ions, or incorporated intopolymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels etc., or incorporated into liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroblasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of a γc-antagonist. A γc-antagonist can be conjugated toantibodies against cell-specific antigens, receptors, ligands, orcoupled to ligands for tissue-specific receptors.

Methods of administrating γc-antagonists of the present embodiments maybe selected as appropriate, depending on factors, such as the type ofdiseases, the condition of subjects, and/or the site to be targeted. Theγc-antagonists can be administered topically, orally, parenterally,rectally, or by inhalation. The term “parenteral” includes subcutaneousinjections, intravenous, intramuscular, intraperitoneal, intracisternalinjection, or infusion techniques. These compositions will typicallyinclude an effective amount of a γc-antagonist, alone or in combinationwith an effective amount of any other active material. Severalnon-limiting routes of administrations are possible includingparenteral, subcutaneous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

The one or more composite peptides disclosed herein can be administeredat any dose, via any of the routes of administration, and at anyfrequency of administration as determined by one of ordinary skill inthe art based on various parameters non-limiting examples of whichinclude the condition being treated, the severity of the condition,patient compliance, efficacy of treatment, side effects, etc.

The amount of the peptide contained in pharmaceutical compositions ofthe present embodiments, dosage form of the pharmaceutical compositions,frequency of administration, and the like may be selected asappropriate, depending on factors, such as the type of diseases, thecondition of subjects, and/or the site to be targeted. Such dosages anddesired drug concentrations contained in the compositions may varyaffected by many parameters, including the intended use, patient's bodyweight and age, and the route of administration. Pilot studies willfirst be conducted using animal studies and the scaling to humanadministration will be performed according to art-accepted practice.

In one embodiment, host cells that have been genetically modified with apolynucleotide encoding at least one γc-antagonist peptide areadministered to a subject to treat a proliferation disorder and/or toreduce the growth of malignant cells. The polynucleotide is expressed bythe host cells, thereby producing the peptides within the subject.Preferably, the host cells are allogeneic or autogeneic to the subject.

In a further aspect, γc-antagonist peptides can be used in combinationwith other therapies, for example, therapies inhibiting cancer cellproliferation and growth. The phrase “combination therapy” embraces theadministration of γc-antagonist peptides and an additional therapeuticagent as part of a specific treatment regimen intended to provide abeneficial effect from the co-action of these therapeutic agents.Administration of these therapeutic agents in combination typically iscarried out over a defined time period (usually minutes, hours, days orweeks depending upon the combination selected).

A combination therapy is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. There therapeutic agents can be administered bythe same route or by different routes. The sequence in which thetherapeutic agents are administered is not narrowly critical.

Combination therapy also can embrace the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different therapeutic agent) and non-drug therapies (such as, butnot limited to, surgery or radiation treatment). Where the combinationtherapy further comprises radiation treatment, the radiation treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andradiation treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the radiation treatment istemporarily removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In certain embodiments, γc-antagonist peptides can be administered incombination with at least one anti-proliferative agent selected from thegroup consisting of chemotherapeutic agent, an antimetabolite, andantitumorgenic agent, and antimitotic agent, and antiviral agent, andantineoplastic agent, an immunotherapeutic agent, and a radiotherapeuticagent.

In certain embodiments, γc-antagonist peptides can be administered incombination with at least one anti-inflammatory agent selected from thegroup consisting of steroids, corticosteroids, and nonsteroidalanti-inflammatory drugs.

Also provided are kits for performing any of the methods providedherein. In some embodiments, kits may include one or more γc-antagonistaccording to any of the embodiments provided herein. In someembodiments, the kit may include instructions. Instructions may be inwritten or pictograph form, or may be on recorded media including audiotape, audio CD, video tape, DVD, CD-ROM, or the like. The kits maycomprise packaging.

Definitions

As used herein, the term “patient” or “subject” refers to the recipientof a any of the embodiments of the composite peptides disclosed hereinand includes all organisms within the kingdom animalia. In someembodiments, any vertebrate including, without limitation, humans andother primates (e.g., chimpanzees and other apes and monkey species),farm animals (e.g., cattle, sheep, pigs, goats and horses), domesticmammals (e.g., dogs and cats), laboratory animals (e.g., rodents such asmice, rats, and guinea pigs), and birds (e.g., domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, etc.) are included. In preferred embodiments, the animal iswithin the family of mammals, such as humans, bovine, ovine, porcine,feline, buffalo, canine, goat, equine, donkey, deer, and primates. Themost preferred animal is human. In some embodiments, the patient is amale or a female.

As used herein, the term “treat” or any variation thereof (e.g.,treatment, treating, etc.), refers to any treatment of a patientdiagnosed with a biological condition, such as CD4−, CD8−, andLGL-leukemia, an autoimmune disease, systemic lupus erythematosis,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, a collagen disease, rheumatoid arthritis,inflammatory bowel disease, diabetes mellitus, psoriasis, a degenerativeneuronal disease, multiple sclerosis, uvietis, inflammation of the eye,graft-versus-host disease (GvHD), myasthenia gravis, Human T-cellLymphotropic type I and II (HTLV-I and HTLV-II)-associated diseases,Adult T-cell Leukemia (ATL), HTLV-associated Myelopathy/Tropical SpasticParaparesis (HAM/TSP), uveitis (HU), arthropathy, pneumopathy,dermatitis, exocrinopathy, myositis, influenza, AIDS, HBV, Herpes,asthma, sinusitis, hay fever, bronchitis, chronic obstructive pulmonarydisease (COPD), allergic rhinitis, acute and chronic otitis, lungfibrosis, NK leukemia/lymphoma and NK-T leukemia/lymphoma.

The term treat, as used herein, includes: (i) preventing or delaying thepresentation of symptoms associated with the biological condition ofinterest in an at-risk patient who has yet to display symptomsassociated with the biological condition; (ii) ameliorating the symptomsassociated with the biological condition of interest in a patientdiagnosed with the biological condition; (iii) preventing, delaying, orameliorating the presentation of symptoms associated with complications,conditions, or diseases associated with the biological condition ofinterest in either an at-risk patient or a patient diagnosed with thebiological condition; (iv) slowing, delaying or halting the progressionof the biological condition; and/or (v) preventing, delaying, slowing,halting or ameliorating the cellular events of inflammation; and/or (vi)preventing, delaying, slowing, halting or ameliorating the histologicalabnormalities and/or other clinical measurements of the biologicalcondition.

The term “symptom(s)” as used herein, refers to common signs orindications that a patient is suffering from a specific condition ordisease.

The term “effective amount,” as used herein, refers to the amountnecessary to elicit the desired biological response. In accordance withthe present embodiments, an effective amount of a γc-antagonist is theamount necessary to provide an observable effect in at least onebiological factor for use in treating a biological condition.

“Recombinant DNA technology” or “recombinant” refers to the use oftechniques and processes for producing specific polypeptides frommicrobial (e.g., bacterial, yeast), invertebrate (insect), mammaliancells or organisms (e.g., transgenic animals or plants) that have beentransformed or transfected with cloned or synthetic DNA sequences toenable biosynthesis of heterologous peptides. Native glycosylationpattern will only be achieved with mammalian cell expression system.Prokaryotic expression systems lack the ability to add glycosylation tothe synthesized proteins. Yeast and insect cells provide a uniqueglycosylation pattern that may be different from the native pattern.

A “nucleotide sequence” refers to a polynucleotide in the form of aseparate fragment or as a component of a larger DNA construct that hasbeen derived from DNA or RNA isolated at least once in substantiallypure form, free of contaminating endogenous materials and in a quantityor concentration enabling identification, manipulation, and recovery ofits component nucleotide sequences by standard molecular biology methods(as outlined in Current Protocols in Molecular Biology).

“Recombinant expression vector” refers to a plasmid comprising atranscriptional unit containing an assembly of (1) a genetic element orelements that have a regulatory role in gene expression includingpromoters and enhances, (2) a structure or coding sequence that encodesthe polypeptide according to the present embodiments, and (3)appropriate transcription and translation initiation sequence and, ifdesired, termination sequences. Structural elements intended for use inyeast and mammalian system preferably include a signal sequence enablingextracellular secretion of translated polypeptides by yeast or mammalianhost cells.

“Recombinant microbial expression system” refers to a substantiallyhomogenous monoculture of suitable microorganisms, for example, bacteriasuch as E. coli, or yeast such as S. cerevisiae, that have stablyintegrated a recombinant transcriptional unit into chromosomal DNA orcarry the recombinant transcriptional unit as a component of a residualplasmid. Generally, host cells constituting a recombinant microbialexpression system are the progeny of a single ancestral transformedcell. Recombinant microbial expression systems will express heterologouspolypeptides upon induction of the regulatory elements linked to astructural nucleotide sequence to be expressed.

As used herein, the section headings are for organizational purposesonly and are not to be construed as limiting the described subjectmatter in any way. All literature and similar materials cited in thisapplication, including but not limited to, patents, patent applications,articles, books, treatises, and internet web pages are expresslyincorporated by reference in their entirety for any purpose. Whendefinitions of terms in incorporated references appear to differ fromthe definitions provided in the present teachings, the definitionprovided in the present teachings shall control. It will be appreciatedthat there is an implied “about” prior to the temperatures,concentrations, times, etc discussed in the present teachings, such thatslight and insubstantial deviations are within the scope of the presentteachings herein.

Although this invention has been disclosed in the context of certainembodiments and examples, those skilled in the art will understand thatthe present invention extends beyond the specifically disclosedembodiments to other alternative embodiments and/or uses of theinvention and obvious modifications and equivalents thereof. Inaddition, while several variations of the invention have been shown anddescribed in detail, other modifications, which are within the scope ofthis invention, will be readily apparent to those of skill in the artbased upon this disclosure.

It is also contemplated that various combinations or sub-combinations ofthe specific features and aspects of the embodiments may be made andstill fall within the scope of the invention. It should be understoodthat various features and aspects of the disclosed embodiments can becombined with, or substituted for, one another in order to form varyingmodes or embodiments of the disclosed invention. Thus, it is intendedthat the scope of the present invention herein disclosed should not belimited by the particular disclosed embodiments described above.

It should be understood, however, that this detailed description, whileindicating preferred embodiments of the invention, is given by way ofillustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art.

EXAMPLES

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

Example 1—Method for Assessing the Inhibitory Activity of γc-AntagonistPeptide

The capacity of any custom derivative peptide prepared according to thepresent embodiments for inhibiting the action of one γc-cytokine familymember is determined using mammalian cellular assays to measure theirproliferative response to the γc-cytokine family member.

For each of the six γc-cytokines, indicator cell lines: NK92, a human NKcell line NK92 available by American Type Culture Collection (ATCC)(catalog #CRL-2407), CTLL-2, a murine CD8 T cells line available fromATCC, and PT-18, a murine mast cell line and its subclone PT-18β, istransfected with human IL-2Rβ gene to make the cells responsive to IL-2and IL-15 (Tagaya et al., 1996, EMBO J. 15:4928-39), and is used toquantitatively determine the γc-cytokine's growth-promoting activity(See Current protocols in Immunology from Wiley and Sons for amethodological reference). The indicator cells demonstrate semi-lineardose-dependent response when measured by a colorimetric WST-1 assay overa range of concentrations (See Clontech PT3946-1 and associated usermanual, incorporated herein by reference, for a detailed description ofthe reagents and methods).

Once the appropriate doses of the cytokine that yield the 50-70% and 95%maximum response from the indicator cell line is determined, variousconcentrations (ranging from 1 μM to 10 μM) of the purified orsynthesized custom derivative peptide is added to each well containingthe cytokine and indicator cells. The reduction in light absorbance at450 nm is used as an indicator of inhibition of cytokine-stimulatedcellular proliferation.

Example 2—The Selective Inhibition of the Growth-Promoting Activities ofCertain γc-Cytokines by BNZ-γ and SEQ ID NO: 3

Using PT-18β cells as described above, the ability of the BNZ-γ peptideto specifically inhibit the growth-promoting activity of selectγc-cytokines was determined (FIG. 3A). IL-3, a non-γc-cytokine thatsupports the growth of PT-18β cells, was used as a negative control.Briefly, PT-18β cells were incubated either with two different dilutionsof BNZ-γ peptide produced by HEK293T cells (1:20 or 1:50 dilution of theoriginal supernatant of HEK293T cells transfected with a BNZ-γexpression construct) or without BNZ-γ peptide in the presence of IL-3,IL-9, IL-15, or IL-4 (1 nM of each cytokine in the culture).

The growth-responses of the cells were determined 2 days after theintroduction of BNZ-γ peptide and the cytokine using the WST-1 assay.The growth-promoting activity of IL-3 (a non γc-cytokine) was notinhibited by BNZ-γ. In contrast, the activity of IL-15 and IL-9 weresignificantly (p<0.01 Student's T test) reduced by the BNZ-γ peptide.Cellular proliferation stimulated by IL-4, another γc-cytokine, was notaffected by the by the addition of BNZ-γ peptide. Results for IL-3,IL-9, IL-15, and IL-4 are shown at FIG. 3A.

In a similar assay, the murine cell line CTTL2 was used. In this assaythe cells were cultured with 0.5 nM of recombinant IL-2 in RPMI 10%fetal Calf Serum. To set up the proliferation assay, cells were washedfrom the cytokines 3 times. Cells were seeded at 1×10(5) cells per wellof a 96-well plate with final concentration of 50 pM of IL-2 or IL-15.Various concentration of BNZ-γ peptide (0.1, 1, and 10 ug/ml) was addedto each well. Cells were cultured for 20 hours and in the last 4 hours,³H-thymidine was added to the plates. Cells were harvested using a platereader. The data are shown in FIG. 3B.

The human NK cell line NK92 was used for a dose response of SEQ ID NO: 3against IL-2, IL-15, and IL-21. Cells were cultured with 0.5 nM ofrecombinant IL-2 in RPMI 10% fetal Calf Serum. To set up theproliferation assay, cells were washed from the cytokines 3 times, andsubsequently seeded at 2.5×10(4) cells per well of a 96-well plate withcytokine concentrations that correspond to 70% NK92 cell proliferationlevels. Commercially purchased SEQ ID NO: 3 (at >99% purity) was addedto each well at a final concentration of 0-10 μM for IL-2, at 0-0.1 μMfor IL-15, and at 0-0.1 μM for IL-21. The growth-responses of the cellswere determined 2 days after the introduction of SEQ ID NO: 3 and thecytokine using the WST-1 assay. Cellular proliferation stimulated by theγc-cytokine IL-2 was not affected by the by the addition of SEQ ID NO: 3up to 10 μM. In contrast, the activity of IL-15 and IL-21 were potentlyinhibited by SEQ ID NO: 3. The data are shown in FIG. 3D.

Example 3—Method for Measuring Inhibition γc-Cytokine Activity byAssaying 3H-Thymidine Incorporation of as a Marker of CellularProliferation

Inhibition of γc-cytokine-induced proliferation of an indicator cellpopulation by antagonist custom derivative peptides is measured by the3H-thymidine incorporation assay. Briefly, radiolabeled thymidine (1microCi) is given to 20-50,000 cells undergoing proliferation in thepresence of cytokines. The cell-incorporated radioactivity is measuredby trapping cell-bound radioactivity to a glass-fiber filter using aconventional harvester machines (for example, Filtermate UniversalHarvester from Perkin-Elmer), after which the radioactivity is measuredusing a b-counter (for example, 1450 Trilux microplate scintillationcounter).

Example 4—Method for Measuring Inhibition γc-Cytokine Activity byAssaying Incorporation of a Cell-Tracker Dye as a Marker of CellularProliferation

Indicator cells are incubated in the presence of a selected γc-cytokineor in the presence of a selected γc-cytokine and a selected customderivative peptide. The cell population is then labeled in vitro using acell-tracker dye, for example, CMFDA, C2925 from Invitrogen, and thedecay of cellular green fluorescence at each cellular division ismonitored using a flow-cytometer (for example, Beckton-DickinsonFACScalibur). Typically, in response to γc-cytokine stimulation 7˜10different peaks corresponding to the number of divisions that the cellshave undergone will appear on the green fluorescence channel. Incubationof the cells with the selected γc-cytokine and antagonist customderivative peptide reduces the number of peaks to only 1 to 3, dependingon the degree of the inhibition.

Example 5—Inhibition of Intracellular Signaling by Custom PeptideDerivative Antagonists

In addition to stimulating cellular proliferation, binding of theγc-cytokines to their receptors causes a diverse array of intracellularevents. (Rochman et al. 2009 Nat. Rev. Immunol. 9:480-90, Pesu et al.2005 Immunol. Rev. 203:127-142.) Immediately after the cytokine binds toits receptor, a tyrosine kinase called Jak3 (Janus-kinase 3) isrecruited to the receptor at the plasma membrane. This kinasephosphorylates the tyrosine residues of multiple proteins including theγc-subunit, STAT5 (Signal Transducer and Activator of Transcription 5)and subunits of the PI3 (Phosphatidylinositol 3) kinase. Among these,the phosphorylation of STAT5 has been implicated in many studies asbeing linked to the proliferation of cells initiated by the γc-cytokine.(Reviewed in Hennighausen and Robinson, 2008 Genes Dev. 22:711-21.) Inaccordance with these published data, whether or not the BNZ-γ peptideinhibits the tyrosine phosphorylation of STAT5 molecule in PT-18β cellsstimulated by IL-15 was examined (results shown in FIG. 4A).

PT-18β cells were stimulated by IL-15 in the presence or absence ofBNZ-γ peptide. Cytoplasmic proteins were extracted from the cellsaccording to a conventional method as described in Tagaya et al. 1996EMBO J. 15:4928-39. The extracted cytoplasmic proteins were resolvedusing a standard SDS-PAGE (Sodium Dodecyl-Sulfate PolyAcrylamide GelElectrophoresis) and the phorphorylation status was confirmed by ananti-phospho-STAT5 antibody (Cell Signaling Technology, Catalog #9354,Danvers Mass.) using immunoblotting (See FIG. 4A, top panel). To confirmthat each lane represented a similar total protein load, the membranewas then stripped, and re-probed with an anti-STAT5 antibody (CellSignaling Technology, Catalog #9358) (See FIG. 4A, bottom panel).

These results demonstrated that tyrosine phosphorylation of STAT5, amarker of signal transduction, was induced by IL-15 in PT-18β cells, andtyrosine phosphorylation of STAT5 was markedly reduced by the BNZ-γpeptide.

Similar to the IL-15 induced tyrosine phosphorylation of STAT5 via Jak3,it is well known in the art that Jak-STAT signaling mediated via IL-21receptor binding preferentially induces Jak-mediated tyrosinephosphorylation of STAT3 (Habib et al. 2003 J Allergy Clin Immunol.112:1033-45). In accordance with these published data, the ability ofSEQ ID NO: 3 to inhibit the tyrosine phosphorylation of the STAT5molecule by IL-2 or IL-15 in CTLL-2 cells, and the ability of SEQ ID NO:3 to inhibit the tyrosine phosphorylation of the STAT3 molecule by IL-21in NK92 cells was examined (results shown in FIG. 4B).

CTLL-2 cells were stimulated by IL-2 or IL-15 in the presence or absenceof SEQ ID NO: 3. CTLL-2 cells were also stimulated by IL-2 or IL-15 inthe presence of anti-IL-2 antibody (R & D Systems, Catalog #MAB202,Minneapolis, Minn.) or anti-IL-15 antibody (R & D Systems, Catalog#MAB247, Minneapolis, Minn.) as positive controls. NK92 cells werestimulated by IL-21 in the presence or absence of SEQ ID NO: 3. NK92cells were also stimulated by IL-21 in the presence of anti-IL-21antibody (Mabtech, Catalog #3540-1-250, Cincinnati, Ohio) as a positivecontrol. Cytoplasmic proteins were extracted from the cells according toa conventional method as described in Tagaya et al. 1996 EMBO J.15:4928-39. The extracted cytoplasmic proteins were resolved using astandard SDS-PAGE (Sodium Dodecyl-Sulfate PolyAcrylamide GelElectrophoresis) and the phosphorylation status was confirmed by ananti-phospho-STAT5 antibody (Cell Signaling Technology, Catalog #9354,Danvers Mass.) or an anti-phospho-STAT3 antibody (Cell SignalingTechnology, Catalog #9145, Danvers Mass.) using immunoblotting (See FIG.4B, top panel). To confirm that each lane represented a similar totalprotein load, the membrane was then stripped, and re-probed with ananti-STAT5 antibody (Cell Signaling Technology, Catalog #9358) or ananti-STAT3 antibody (Cell Signaling Technology, Catalog #9139) (See FIG.4B, bottom panel).

Example 6—Rational Design for BNZ-γ Derivative Antagonistic Peptides

Derivative peptides are prepared based from the core sequenceD/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO: 2) (where X denotes anyamino acid) by substituting the defined amino acids of the core sequencewith amino acids having identical physico-chemical properties asdesignated in FIG. 2.

Alternatively, custom peptides or their derivative peptides can beprepared based on the sequence alignment of the D-helix regions ofdifferent γc-cytokine family members. For example, as shown in FIG. 6,one or more sequences conserved in γc-cytokine family (SEQ ID NO: 4-SEQID NO: 9) members can be combined to form a peptide such as SEQ ID NO:3.

Example 7—Method of Identifying the Inhibitory Specificity ofAntagonistic Custom Derivative Peptides

The γc-cytokine inhibitory specificity of antagonistic custom derivativepeptides is determined by assaying the ability of a custom derivativepeptide to inhibit the proliferative response of a cytokine-responsivecell line to each of the 6 γc-cytokines. For example, a mouse cell line,CTLL-2, is used to determine if a candidate peptide inhibits thefunction of IL-2 and IL-15. PT-18(β) cells are used to determine if acandidate peptide inhibits the function of IL-4 and IL-9. PT-18 (7a)cells are used to determine if a candidate peptide inhibits the functionof IL-7, and PT-18(21a) cells are used to determine if a candidatepeptide inhibits the function of IL-21. PT-18(β) denotes a subclone ofPT-18 cells that exogenously express human IL-2Rβ by gene transfection(See Tagaya et al. 1996), PT-18(7a) denotes a subclone that expresseshuman IL-7Ra by gene transfection and PT-18(21Ra) cells express humanIL-21Ra.

Another alternative is to use other cell lines that respond to an arrayof cytokines. An example of this cell line in a human NK cell line NK92that is commercially available by ATCC (catalog #CRL-2407). This cellline is an IL-2 dependent cell line that responds to other cytokinesincluding IL-9, IL-7, IL-15, IL-12, IL-18, IL-21 (Gong et al. 1994Leukemia 8: 652-658, Kingemann et al., 1996, Biol Blood MarrowTransplant 2:68; 75, Hodge D L et al., 2002 J. Immunol. 168:9090-8).

The human NK cell line NK92 was used to determine the γc-cytokineinhibitory specificity of SEQ ID NO: 3. In this assay the cells werecultured with 0.5 nM of recombinant IL-2 in RPMI 10% fetal Calf Serum.To set up the inhibitory specificity assay, cells were washed from thecytokines 3 times. Cells were seeded at 2.5×10(4) cells per well of a96-well plate with cytokine concentrations that correspond to 70% NK92cell proliferation levels. Commercially purchased SEQ ID NO: 3 (at 70%purity levels) was added to each well at a final concentration of 2.5μM. The growth-responses of the cells were determined 2 days after theintroduction of SEQ ID NO: 3 and the cytokine using the WST-1 assay.Cellular proliferation stimulated by the γc-cytokines IL-2, IL-4, andIL-9 was not affected by the by the addition of SEQ ID NO: 3. Incontrast, the activity of IL-15 and IL-21 were significantly reduced bySEQ ID NO: 3. Results for IL-2, IL-4, IL-9, IL-15, and IL-21 are shownat FIG. 3C.

Example 8—Enhanced Inhibitory Activity of Custom Peptide DerivativesContaining Intra-Peptide Hydrocarbon Linker Element

The human NK cell line NK92 was used to determine the enhancedinhibitory activity of custom peptide derivatives containingintra-peptide hydrocarbon linker element. Cells were cultured with 0.5nM of recombinant IL-2 in RPMI 10% fetal Calf Serum. To set up theproliferation assay, cells were washed from the cytokines 3 times. Cellswere seeded at 2.5×10(4) cells per well of a 96-well plate with a finalIL-15 concentration of 0.25 ng/mL. Commercially purchased unmodified SEQID NO: 3 and custom peptide derivatives of SEQ ID NO: 3 containing anintra-peptide hydrocarbon linker element were added to each well atequal concentrations. The growth-responses of the cells were determined2 days after the introduction of the peptides and the cytokine using theWST-1 assay. Multiple custom peptide derivatives of SEQ ID NO: 3containing an intra-peptide hydrocarbon linker element showed enhancedinhibitory activity as compared to the unmodified SEQ ID NO: 3. The dataare shown in FIG. 7.

Example 9—Time-Course Protease Stability Measurement of Custom PeptideDerivatives Containing Intra-Peptide Hydrocarbon Linker Element inSimulated Intestinal Fluid

To evaluate the gastric stability of the custom peptide derivativescontaining one or more intra-peptide hydrocarbon linker elements of thepresent embodiments a time-course protease stability measurement ofunmodified SEQ ID NO: 3 in comparison with a representative custompeptide derivative of SEQ ID NO: 3 containing one hydrocarbon linkerelement (SEQ ID NO: 39), and another representative custom peptidederivative of SEQ ID NO: 3 containing two hydrocarbon linker elementsand certain amino acid positions in the (D) stereochemical configuration(SEQ ID NO: 57) was carried out in simulated intestinal fluid over 60minutes. All peptides were commercially synthesized and purchased.Simulated intestinal fluid was made according to USP specifications(Test Solutions, United States Pharmacopeia 36). 50 μL of 10 mg/mLpeptide was mixed with 450 μL pre-warmed (37° C.) simulated intestinalfluid, and aliquots of the reaction mixture were removed at prescribedtime intervals over a time-period of 60 minutes and stopped with 0.1 MHCl. Peptide stability was measured by reversed phase HPLC on aPhenomenex Aeris Peptide column (4.6×250 mm) with settings: 3.6 μmparticle size, non-porous; 5.0 μL injection, 0.5 mL/min; mobile phase A:25% Acetonitrile with 0.1% TFA; mobile phase B: 100% Acetonitrile with0.1% TFA. The multi-step buffer gradient goes from 100% A to 100% B over26 minutes. Time point 0 minutes was set to 100% undigested peptide, anddata at later time-points were graphed and connected via a smooth curveout to the end-point of 60 minutes (FIG. 8A). Results show thepercentage of undigested peptide present in the simulated intestinalfluid is greatly enhanced with one or more hydrocarbon linker elementsand certain amino acid positions in the (D) stereochemicalconfiguration, compared to the unmodified peptide. The data are shown inFIG. 8A.

Example 10—Preparation of γc-Antagonist Peptides

Custom derivative γc-antagonist peptides are synthesized chemically bymanual and automated processes.

Manual synthesis: Classical liquid-phase synthesis is employed, whichinvolves coupling the carboxyl group or C-terminus of one amino acid tothe amino group or N-terminus of another. Alternatively, solid-phasepeptide synthesis (SPPS) is utilized.

Automated synthesis: Many commercial companies provide automated peptidesynthesis for a cost. These companies use various commercial peptidesynthesizers, including synthesizers provided by Applied Biosystems(ABI). Custom derivative γc-antagonist peptides are synthesized byautomated peptide synthesizers.

Example 11—Biological Production of Custom Derivative γc-AntagonistPeptides Using Recombinant Technology

A custom derivative γc-antagonist peptide is synthesized biologically asa pro-peptide that consists of an appropriate tagging peptide, a signalpeptide, or a peptide derived from a known human protein that enhancesor stabilizes the structure of the BNZ-γ peptide or a peptide comprisingthe sequence of SEQ ID NO: 3 or a derivative thereof, and improves theirbiological activities. If desired, an appropriate enzyme-cleavagesequence proceeding to the N-terminus of the peptide shall be designedto remove the tag or any part of the peptide from the final protein.

A nucleotide sequence encoding the custom derivative peptide with a stopcodon at the 3′ end is inserted into a commercial vector with a tagportion derived from thioredoxin of E. coli and a special peptidesequence that is recognized and digested by an appropriate proteolyticenzyme (for example, enterokinase) intervening between the tag portionand the nucleotide sequence encoding the custom derivative peptide andstop codon. One example of a suitable vector is the pThioHis plasmidavailable from Invitrogen, CA. Other expression vectors may be used.

Example 12—Conjugation of Custom Peptides and Derivative to CarrierProteins for Immunization Purposes and Generation of Antibody Againstthe Custom Peptides

BNZ-γ and other custom derivative peptides, such as a peptide comprisingthe sequence of SEQ ID NO:3 or a derivative thereof are used to immunizeanimals to obtain polyclonal and monoclonal antibodies. Peptides areconjugated to the N- or the C-terminus of appropriate carrier proteins(for example, bovine serum albumin, Keyhole Limpet Hemocyanin (KLH),etc.) by conventional methods using Glutaraldehyde orm-Maleimidobenzoyl-N-Hydroxysuccinimide Ester. The conjugated peptidesin conjunction with an appropriate adjuvant are then used to immunizeanimals such as rabbits, rodents, or donkeys. The resultant antibodiesare examined for specificity using conventional methods. If theresultant antibodies react with the immunogenic peptide, they are thentested for the ability to inhibit individual γc-cytokine activityaccording to the cellular proliferation assays described in Examples1-3. Due to the composite nature of the derivative peptides it ispossible to generate a single antibody that recognizes two differentcytokines simultaneously, because of the composite nature of thesepeptides.

Example 13—Method for Large Scale Production of Custom Derivativeγc-Antagonist Peptides

Recombinant proteins are produced in large scale by the use of cell-freesystem as described elsewhere. (See Takai et al., 2010 Curr. Pharm.Biotechnol. 11(3):272-8.) Briefly, cDNAs encoding the γc-antagonistpeptide and a tag are subcloned into an appropriate vector (See Takai etal., 2010 Curr. Pharm. Biotechnol. 11(3):272-8), which is subjected toin vitro transcription, followed immediately by an in vitro translationto produce the tagged peptide. The pro-polypeptide is then purifiedusing an immobilized antibody recognizing the tagged epitope, treated bythe proteolytic enzyme and the eluate (which mostly contains the customderivative peptide of interest) is tested for purity using conventional18% Tricine-SDS-PAGE (Invitrogen) and conventional comassie staining.Should the desired purity of the peptide not be met (>98%), the mixtureis subjected to conventional HPLC (high-performance liquidchromatography) for further purification.

Example 14—Use of Custom Derivative γc-Antagonist Peptides to BlockCytokine Function in HAM/TSP

HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) isa chronic progressive myelopathy seen in some people infected with HumanT-Lymphotropic Virus Type I (HTLV-I). Infiltration of lymphocytes in thespinal cord is associated with the immune response to HTLV-I and resultsin the release of certain cytokines. Some of these cytokines may alsodamage nerves.

Patients with HAM/TSP show an elevated state of the immune system thatis similar to that observed in autoimmune diseases (Oh et al. 2008Neurol Clin. 26:781-785). This elevated state is demonstrated by theability of HAM/TSP patient's T-cells to undergo spontaneousproliferation in an ex vivo culture for about a week in the absence ofexogenously added cytokines. The spontaneous proliferation of T-cells inHAM/TSP patients is attributed, at least partly, to autocrine/paracrineloops of IL-2, IL-9, and IL-15. It has been shown that adding blockingantibody against the IL-2 or IL-15 receptors can inhibit spontaneousT-cell proliferation in a HAM/TSP ex vivo culture system.

These observations, along with other data derived from ex vivo studies,have provided the rationale for taking two monoclonal antibodies (ananti-IL-2 receptor alpha or anti-Tac and an anti-IL-15 receptor betachain) into the clinic for treatment of HAM/TSP (Azimi et al. 2001 Proc.Natl. Acad. Sci. 98:14559-64., Azimi et al., 1999 J. Immunol163:4064-72). Anti-cytokine receptor antagonists according to theembodiments described herein, would not only be valuable as atherapeutic immuno-modulatory agent for treatment of HAM/TSP, butmodulation of immune response in HAM/TSP by anti-cytokine receptorantagonists according to the present embodiments acts proof-of-conceptfor the use of the anti-cytokine receptor antagonists according to thepresent embodiments in the treatment of other auto-immune diseases.

To demonstrate the efficacy of custom derivative γc-antagonist peptidesaccording to the embodiments described herein, we tested the ability ofBNZ-γ peptide to block immune response to HTLV-I in a spontaneous T-cellproliferation assay using a HAM/TSP ex vivo culture system.Proliferation assays were performed on HAM/TSP patient blood sampleswith and without the addition of BNZ-γ. These assays evaluated theability of BNZ-γ to block the function of cytokines, such as IL-2 andIL-15, present in the ex vivo HAM/TSP patient blood culture and preventspontaneous T-cell proliferation in these samples.

In an ex vivo spontaneous T-cell proliferation assay, PBMC from HAM/TSPpatient was cultured at 1×10(6) cells per well of a 96 well plate inRPMI-10% FCS. Increasing concentrations of BNZ-γ peptide were added toeach well. As a control, an irrelevant peptide was used in similarfashion. The cells were incubated in a 37° C. CO2 incubator for 3, 4,and 6 days. The amount of 1 uCi of ³H-thymidine was added to the cells.After an additional 6 hour incubation, cells were harvested theirproliferation rate was measured. The data for a representative HAM/TSPpatient is shown in FIG. 5A-FIG. 5D. As indicated in FIG. 5A-FIG. 5D,BNZ-γ peptide inhibits the spontaneous proliferation of T-cells inHAM/TSP culture at a concentration of about 1 ug/ml.

Other immunological markers were additionally measured in this assay.The percentage of the viral specific CD8 cells was measured during theex vivo culture using viral protein tetramers. The population ofCD4+CD25+ cells, a marker of T-cell activation, as well as Ki67staining, a marker of T-cell proliferation, was monitored in a flowcytometry assay.

Other forms of the conjugated BNZ-γ peptide derivative or a custompeptide comprising the sequence of SEQ ID NO: 3, and a derivativethereof can be used in a similar future assay. They include albumin,BSA, PEG that can be conjugated to the peptide after chemical synthesis.Other biological forms of custom peptides such as the BNZ-γ peptideconjugate or a custom peptide comprising the sequence of SEQ ID NO: 3,and a derivative thereof may include regions of known protein entities(including but not limited to Fc region of human IgG) that are fused tothe custom peptides.

Example 15—Method of Treating Adult T-Cell Leukemia (ATL) in a HumanPatient by Administration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from Adult T-cell Leukemia is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, BNZ-γ, a custom peptide comprisingthe sequence of SEQ ID NO: 3, or a derivative thereof is administered tothe patient for a period of time determined by the physician. Treatmentis determined to be effective if patient enters remission.

Example 16—Method of Treating HAM/TSP in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

A human patient suffering from HAM/TSP is identified. An effective dose,as determined by the physician, of custom derivative γc-antagonistpeptide, for example, BNZ-γ, a custom peptide comprising the sequence ofSEQ ID NO: 3, or a derivative thereof is administered to the patient fora period of time determined by the physician. Treatment is determined tobe effective if patient's symptoms improve or if the progression of thedisease has been stopped or slowed down.

Example 17—Use of Custom Derivative γc-Antagonist Peptides to BlockCytokine Function

A human patient suffering a disease state who is in need of reducing thefunction of at least IL-15 and IL-21 is identified. An effective dose,as determined by the physician, of custom derivative γc-antagonistpeptide, for example, a composite peptide comprising the sequence of SEQID NO:3 or a derivative thereof is administered to the patient for aperiod of time determined by the physician. Treatment is determined tobe effective if patient's symptoms improve or if the progression of thedisease has been stopped or slowed down.

Example 18—Method of Treating Celiac Disease in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

In celiac disease IL-15 is chronically up-regulated in the laminapropria and epithelium of the intestine and directly correlates with theseverity of mucosal damage (Jabri et al. 2000 Gastroenterology118:867-879., Maiuri et al. 2000 Gastroenterology 119:996-1006., Mentionet al. 2003 Gastroenterology 125:730-45.; Di Sabatino et al. 2006 Gut55:469-77.), and IL-21 production and function plays a positive role fordisease progression (De Nitto et al. 2009 World J Gastroenterol15:4609-14.). IL-15 has also been shown to directly correlate, andpositively regulate, the increased expression of IL-21 observed in theCD mucosal environment (Sarra et al. 2013 Mucosal Immunol 6: 244-55.). Arecent study also identified synergistic effects of IL-2, IL-15 andIL-21 contribute greatly to the pathogenesis of refractory CD(Kooy-Winkelaar, et al., 2017 Proc Natl Acad Sci USA 114: E980-9.).

A human patient suffering from Celiac disease is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, BNZ-γ, a custom peptide comprisingthe sequence of SEQ ID NO: 3, or a derivative thereof is administered tothe patient for a period of time determined by the physician. Treatmentis determined to be effective if patient's symptoms improve or if theprogression of the disease has been stopped or slowed down.

Example 19—Method of Treating Rheumatoid Arthritis in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

In rheumatoid arthritis (RA) IL-15 expression is elevated in thesynovial fluid and rheumatoid nodules of patients, increases thetrans-endothelial migration of CD4+ and CD8+ T cells in the RAenvironment, and serum levels of IL-15 are positively correlated withdisease progression (Oppenheimer-Marks et al. 1998 J Clin Invest101:1261-72., Harada et al. 1999 Arthritis Rheum 42:1508-16.,Gonzalez-Alvaro et al. 2003 Clin Exp Rheumatol 21:639-42., Hessian etal. 2003 Arthritis Rheum 48:334-8., Ruckert et al. 2009 Immunology126:63-73.). In RA IL-21 positively impacts the expression ofpro-inflammatory cytokines TNF-α and IL-6, and the invasion andmigration of fibroblast-like synoviocytes (FLS). FLS play a key role inRA pathogenesis through aggressive proliferation and invasion inpatients (Xing et al. 2016 Clin Exp Immunol 184:147-58., Xing et al.2016 Scand J Immunol 83:64-71.).

A human patient suffering from rheumatoid arthritis is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, BNZ-γ, a custom peptide comprisingthe sequence of SEQ ID NO: 3, or a derivative thereof is administered tothe patient for a period of time determined by the physician. Treatmentis determined to be effective if patient's symptoms improve or if theprogression of the disease has been stopped or slowed down.

Example 19—Method of Treating Multiple Sclerosis in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

In multiple sclerosis (MS) IL-15 expression is elevated in the serum andcerebrospinal fluid of patients (Kivisakk et al. 1998 Clin Exp Immunol111:193-7., Pashenkov et al. 1999 Scand J Immunol 50:302-8.,Vaknin-Dembinsky et al. 2008 J Neuroimmunol 195:135-9.). Brain lesionsof MS patients also contain astrocytes and B cells with increased levelsof IL-15 (Saikali et al. 2010 J Immunol 185:5693-703., Schneider et al.2011 J Immunol 187:4119-28.). In MS IL-21 production plays a positiverole for disease progression and its expression is strongly elevated inCD4+ cells of acute and chronic MS lesions (Tzartos et al. 2011 Am JPathol 178:794-802., Ghalamfarsa et al. 2016 J Immunotoxicol13:274-85.).

A human patient suffering from multiple sclerosis is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, a composite peptide comprising thesequence of SEQ ID NO:3 or a derivative thereof is administered to thepatient for a period of time determined by the physician. Treatment isdetermined to be effective if patient's symptoms improve or if theprogression of the disease has been stopped or slowed down.

Example 20—Method of Treating Type 1 Diabetes Mellitus in a HumanPatient by Administration of Custom Derivative γc-Antagonist Peptide

In type 1 diabetes mellitus (T1D) IL-15 expression is elevated in theserum of patients (Kuczynski et al. 2005 Diabetes Res Clin Pract69:231-6.). T1D could be completely prevented by the inhibition of IL-15signaling at the onset of insulitis in the non-obese diabetic mousemodel (Bobbala et al. 2012 Diabetologia 55: 3010-3020.). The humanpancreatic islet expression of IL-15 is also observed in T1D patients(Chen et al. 2013 Proc Natl Acad Sci USA 110:13534-9.). Furthermore,high IL-21 production is correlated with T1D disease progression(Ferreira et al. 2015 Diabetologia 58: 781-90.).

A human patient suffering from type 1 diabetes mellitus is identified.An effective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, a composite peptide comprising thesequence of SEQ ID NO:3 or a derivative thereof is administered to thepatient for a period of time determined by the physician. Treatment isdetermined to be effective if patient's symptoms improve or if theprogression of the disease has been stopped or slowed down.

Example 21—Method of Treating Psoriasis in a Human Patient byAdministration of Custom Derivative γc-Antagonist Peptide

The expression of IL-15 is elevated in skin lesions in psoriasispatients (Waldmann 2013 J Investig Dermatol Symp Proc 16:528-30.). IL-21production and function plays a positive role for psoriasis diseaseprogression (Caruso et al. 2009 Cell Cycle 8: 3629-30., Botti et al.2012 Curr Pharm Biotechnol 13: 1861-7.), and expression of theγc-cytokine is elevated in the serum of patients and is associated withdisease severity (He et al. 2012 Br J Dermatol 167: 191-3.).

A human patient suffering from psoriasis is identified. An effectivedose, as determined by the physician, of custom derivative γc-antagonistpeptide, for example, a composite peptide comprising the sequence of SEQID NO:3 or a derivative thereof is administered to the patient for aperiod of time determined by the physician. Treatment is determined tobe effective if patient's symptoms improve or if the progression of thedisease has been stopped or slowed down.

Example 22—Method of Treating Inflammatory Bowel Diseases in a HumanPatient by Administration of Custom Derivative γc-Antagonist Peptide

In inflammatory bowel diseases (IBD, including ulcerative colitis andCrohn's disease) increased levels of IL-15 is observed in inflamedmucosa (Liu et al. 2000 J Immunol 164:3608-15., Vainer et al. 2000Cytokine 12:1531-6.). IBD patients also experience increased levels ofIL-21 in the gut (Monteleone et al. 2005 Gastroenterology 128: 687-94.),which is positively correlated with increased gut mucosa inflammation(De Nitto et al. 2010 World J Gastroenterol 16: 3638-41.).

A human patient suffering from an inflammatory bowel disease isidentified. An effective dose, as determined by the physician, of customderivative γc-antagonist peptide, for example, a composite peptidecomprising the sequence of SEQ ID NO:3 or a derivative thereof isadministered to the patient for a period of time determined by thephysician. Treatment is determined to be effective if patient's symptomsimprove or if the progression of the disease has been stopped or sloweddown.

Example 23—Method of Treating Systemic Lupus Erythematosus in a HumanPatient by Administration of Custom Derivative γc-Antagonist Peptide

In systemic lupus erythematosus (SLE) IL-15 expression is elevated inthe serum of patients (Aringer et al. 2001 Rheumatology (Oxford)40:876-81.). Dysfunction of IL-15 signaling is positively associatedwith SLE pathogenesis (Baranda et al. 2005 Rheumatology (Oxford)44:1507-13.). In SLE IL-21 expression is elevated in the serum ofpatients (Nakou et al. 2013 Clin Exp Rheumatol 31:172-9.). Geneticpolymorphisms in IL-21 are also positively associated with SLE (Sawalhaet al. 2008 Ann Rheum Dis 67:458-61.). IL-21 production in SLE ispositively correlated to T cell and B cell alterations observed in SLEpathogenesis (Terrier et al. 2012 J Rheumatol 39:1819-28.), and IL-21signaling is critical for SLE pathogenic progression in the BXSB-Yaamurine disease model (Bubier et al. 2009 Proc Natl Acad Sci USA 106:1518-23.).

A human patient suffering from systemic lupus erythematosus isidentified. An effective dose, as determined by the physician, of customderivative γc-antagonist peptide, for example, a composite peptidecomprising the sequence of SEQ ID NO:3 or a derivative thereof isadministered to the patient for a period of time determined by thephysician. Treatment is determined to be effective if patient's symptomsimprove or if the progression of the disease has been stopped or sloweddown.

Example 24—Method of Treating Alopecia Areata Disease in a Human Patientby Administration of Custom Derivative γc-Antagonist Peptide

In alopecia areata disease (AAD) IL-15 expression is elevated in thelesional scalp biopsies of patients (Fuentes-Duculan et al. 2016 ExpDermatol 4:282-6., Waldmann 2013 J Investig Dermatol Symp Proc16:S28-30.), and antibodies targeting the γc-cytokines IL-2 and IL-15each showed inhibitory activity in an AAD mouse model, but none of theblocking antibodies alone could reverse established AAD (Xing et al.2014 Nat Med 9:1043-9.). In AAD IL-21 expression is elevated in theserum of patients versus healthy controls (Atwa et al. 2016 Int JDermatol 55:666-72.). Genome-wide association studies have alsopositively correlated IL-2 and IL-21 with AAD (Jagielska et al. 2012 JInvest Dermatol 132:2192-7, Petukhova et al. 2010 Nature 466:113-7.).

A human patient suffering from alopecia areata disease is identified. Aneffective dose, as determined by the physician, of custom derivativeγc-antagonist peptide, for example, BNZ-γ, a custom peptide comprisingthe sequence of SEQ ID NO: 3, or a derivative thereof is administered tothe patient for a period of time determined by the physician. Treatmentis determined to be effective if patient's symptoms improve or if theprogression of the disease has been stopped or slowed down.

Example 25—Time-Course Protease Stability Measurement of Custom PeptideDerivatives Containing Intra-Peptide Hydrocarbon Linker Element inSimulated Intestinal Fluid

To evaluate the gastric stability of the custom peptide derivativescontaining one or more intra-peptide hydrocarbon linker elements of thepresent embodiments a time-course protease stability measurement ofunmodified SEQ ID NO: 3 in comparison with a representative custompeptide derivative of SEQ ID NO: 3 containing one hydrocarbon linkerelement (SEQ ID NO: 39), and another representative custom peptidederivative of SEQ ID NO: 3 containing two hydrocarbon linker elementsand certain amino acid positions in the (D) stereochemical configuration(SEQ ID NO: 57) was carried out in simulated intestinal fluid over 60minutes. An additional time-course protease stability experiment wasconducted in simulated intestinal fluid over 120 minutes to compare tworepresentative custom peptide derivatives of SEQ ID NO: 3, the firstcontaining one hydrocarbon linker element (SEQ ID NO: 39), and thesecond containing two hydrocarbon linker elements (SEQ ID NO: 83). Allpeptides were commercially synthesized and purchased. Simulatedintestinal fluid was made according to USP specifications (TestSolutions, United States Pharmacopeia 36). 50 μL of 10 mg/mL peptide wasmixed with 450 μL pre-warmed (37° C.) simulated intestinal fluid, andaliquots of the reaction mixture were removed at prescribed timeintervals over a time-period of 60 minutes and stopped with 0.1 M HCl.Peptide stability was measured by reversed phase HPLC on a PhenomenexAeris Peptide column (4.6×250 mm) with settings: 3.6 μm particle size,non-porous; 5.0 μL injection, 0.5 mL/min; mobile phase A: 25%Acetonitrile with 0.1% TFA; mobile phase B: 100% Acetonitrile with 0.1%TFA. The multi-step buffer gradient goes from 100% A to 100% B over 26minutes. Time point 0 minutes was set to 100% undigested peptide, anddata at later time-points were graphed and connected via a smooth curveout to the end-point of 60 minutes (FIG. 8A) or 120 minutes (FIG. 8B).Results show the percentage of undigested peptide present in thesimulated intestinal fluid is greatly enhanced with one or morehydrocarbon linker elements and certain amino acid positions in the (D)stereochemical configuration. The data are shown in FIG. 8A and FIG. 8B.

Example 26—Inhibition of Cytokine-Induced Gene Transcription byRepresentative Custom Peptide Derivative

The cytokine interferon gamma (IFNγ) represents a useful marker forinflammation in multiple human immune-inflammatory disease states.Transcription of the IFNγ gene is inducible by IL-15, IL-21, the non-γccytokine IL-12, and other select cytokines. The ability to blockcytokine-induced IFNγ transcription therefore represents another usefulapproach to determine the biological activity of custom peptidederivatives.

NK92 cells were brought to a quiescent state by PBS washing andculturing in the absence of cytokine for 48 hours. Cells were treatedwith SEQ ID NO: 83 at M for 10 minutes, and then stimulated with humanIL-15, IL-21, or IL-12 at 1 ng/mL for 2 hours. A cytokine only treatmentgroup, and another treatment group where the NK92 cells were leftuntreated by peptide, and not stimulated by cytokine were also includedas control conditions. Following incubation with peptide and cytokine,cells were harvested, RNA was isolated for cDNA generation and used astemplate for qPCR analysis to quantify IFNγ transcripts. SEQ ID NO: 83showed inhibition of IL-15 and IL-21 induction of IFNγ genetranscription, but did not block the non-γc cytokine IL-12 induction ofIFNγ gene transcription. The data are shown in FIG. 9.

REFERENCES

All references cited in this disclosure are incorporated herein byreference in their entireties.

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1.-80. (canceled)
 81. A composite peptide comprising amino acidsequences of at least two interleukin (IL) protein gamma-c-box D-helixregions, wherein the composite peptide comprises an amino acid sequencehaving at least 50% identity to the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), wherein thecomposite peptide comprises at least one hydrocarbon linker element. 82.The composite peptide of claim 81, wherein the composite peptidecomprises at least two alpha-alkenyl substituted amino acids.
 83. Thecomposite peptide of claim 82, wherein the at least two alpha-alkenylsubstituted amino acids are selected from the group consisting ofR-propenylalanine (CAS: 288617-76-5; R3Ala), S-propenylalanine (CAS:288617-71-0; S3Ala), D-allylglycine (CAS: 170642-28-1; D3Gly),L-allylglycine (CAS: 146549-21-5; L3Gly), R-pentenylalanine (CAS:288617-77-6; R5Ala), S-pentenylalanine (CAS: 288617-73-2; S5Ala),R-pentenylglycine (CAS: 1093645-21-6; R5Gly), S-pentenylglycine (CAS:856412-22-1; S5Gly), R-butenylalanine (CAS: 1311933-82-0; R4Ala),S-butenylalanine (CAS: 288617-72-1; S4Ala), R-butenylglycine (CAS:865352-21-2; R4Gly), S-butenylglycine (CAS: 851909-08-5; S4Gly),R-hexenylalanine (CAS: 288617-78-7; R6Ala), S-hexenylalanine (CAS:288617-74-3; S6Ala), R-hexenylglycine (CAS: 1208226-88-3; R6Gly),S-hexenylglycine (CAS: 1251904-51-4; S6Gly), R-heptenylalanine (CAS:1311933-84-2; R7Ala), S-heptenylalanine (CAS: 1311933-83-1; S7Ala),R-heptenylglycine (CAS: 1262886-63-4; R7Gly), S-heptenylglycine (CAS:1058705-57-9; S7Gly), di-substituted bis-propenylglycine (CAS:1311992-97-8; bis3Gly), di-substituted bis-pentenylglycine (CAS:1068435-19-7; bis5Gly), di-substituted bis-butenylglycine (bis4Gly),di-substituted bis-hexenylglycine (bis6Gly), di-substitutedbis-heptenylglycine (bis7Gly), R-octenylalanine (CAS: 945212-26-0;R8Ala), S-octenylalanine (CAS: 288617-75-4; S8Ala), R-octenylglycine(CAS: 1191429-20-5; R8Gly), and S-octenylglycine (CAS: 1262886-64-5;S8Gly).
 84. The composite peptide of claim 82, wherein the at least twoalpha-alkenyl substituted amino acids are separated by three amino acidswithin the amino acid sequence and is linked by the at least onehydrocarbon linker element spanning a single α-helical turn of thecomposite peptide.
 85. The composite peptide of claim 81, wherein thecomposite peptide is configured to inhibit the activity of one or more7c-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21.
 86. The composite peptide of claim 81, whereinthe composite peptide comprises an amino acid sequence of at least 70%identity to the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3).
 87. Thecomposite peptide of claim 81, wherein the composite peptide comprisesthe amino acid sequence P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQID NO: 3).
 88. The composite peptide of claim 81, wherein the compositepeptide is configured to inhibit the activity of IL-15, IL-21, or acombination thereof.
 89. The composite peptide of claim 81, wherein agastric stability of the composite peptide is enhanced compared to anunmodified composite peptide without a hydrocarbon linker element. 90.The composite peptide of claim 81, wherein the at least one hydrocarbonlinker element comprises one or more covalently attached chemical groupsubstituents, wherein the chemical group substituents are selected fromthe group consisting of alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, haloalkyl, halo, oxo, nitro, hydroxy, mercapto,carboxy, alkylcarbonyl, alkoxycarbonyl, alkanesulfonyl, amino, amido,azido, cyano, PEG, affinity labels, targeting moiety, fatty-acid derivedacyl group, biotin, radioisotopes, therapeutic agents, non-proteinfluorescent chemical groups, and protein fluorescent groups.
 91. Thecomposite peptide of claim 81, wherein the hydrocarbon linker element isan intra-peptide linker element.
 92. The composite peptide of claim 81,wherein amino acid sequence of SEQ ID NO: 3 comprises an amino acidsequence represented by SEQ ID NO:
 83. 93. A pharmaceutical compositioncomprising: a therapeutically effective amount of a composite peptide;and a pharmaceutically acceptable carrier, diluent, excipient orcombination thereof; wherein the composite peptide comprises an aminoacid sequence having at least 50% identity to the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), and whereinthe composite peptide comprises at least one hydrocarbon linker element.94. The pharmaceutical composition of claim 93, wherein the compositepeptide is configured to modulate the activity of one or more7c-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21.
 95. A method of preventing or treating a Tccytokine-mediated disease, an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease, aninflammatory respiratory disease, or a cosmetic condition, the methodcomprising: providing a pharmaceutical composition comprising: atherapeutically effective amount of a composite peptide; and apharmaceutically acceptable carrier, diluent, excipient or combinationthereof; wherein the composite peptide comprises an amino acid sequencehaving at least 50% identity to the amino acid sequenceP-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ ID NO: 3), wherein thecomposite peptide comprises at least one hydrocarbon linker element,wherein the composite peptide modulates the activity of one or more7c-cytokines selected from the group consisting of IL-2, IL-4, IL-7,IL-9, IL-15, and IL-21.
 96. The method of claim 95, wherein the γccytokine-mediated disease is selected from the group consisting of CD4leukemia, CD8 leukemia, LGL leukemia, systemic lupus erythematosis,Sjögren's syndrome, Wegener's granulomatosis, Celiac disease,Hashimoto's thyroiditis, rheumatoid arthritis, diabetes mellitus,psoriasis, multiple sclerosis, uvietis, inflammation of the eye,graft-versus-host disease (GvHD), inflammatory bowel disease, ulcerativecolitis, Crohn's disease, Systemic Lupus Erythematosus, and alopeciaareata.
 97. The method of claim 95, wherein the HAM/TSP associateddisease is selected from the group consisting of Adult T-cell Leukemia(ATL), HTLV-associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP), and other non-neoplastic inflammatory diseases associatedwith HTLV such as uveitis (HU), arthropathy, pneumopathy, dermatitis,exocrinopathy, and myositis.
 98. The method of claim 95, wherein theinflammatory respiratory disease is selected from the group consistingof asthma, sinusitis, hay fever, bronchitis, chronic obstructivepulmonary disease (COPD), allergic rhinitis, acute and chronic otitis,and lung fibrosis.
 99. The method of claim 95, wherein the wherein thecosmetic condition is selected from the group consisting of acne, hairloss, sunburn, nail maintenance, and appearance of aging.
 100. A kit forpreventing or treating a condition in a patient, wherein the conditionis a γc cytokine-mediated disease, an HTLV-1-associated myelopathy(HAM)/tropical spastic paraparesis (TSP) associated disease, aninflammatory respiratory disease, a cosmetic condition, or a combinationthereof, the kit comprising a pharmaceutical composition, wherein thepharmaceutical composition comprises: a therapeutically effective amountof a composite peptide; and a pharmaceutically acceptable carrier,diluent, excipient or combination thereof; wherein the compositecomprises an amino acid sequence having at least 50% identity to theamino acid sequence P-K-E-F-L-E-R-F-V-H-L-V-Q-M-F-I-H-Q-S-L-S (SEQ IDNO: 3), wherein the composite peptide comprises at least one hydrocarbonlinker element, wherein the composite peptide modulates the activity ofone or more 7c-cytokines selected from the group consisting of IL-2,IL-4, IL-7, IL-9, IL-15, and IL-21.
 101. The kit of claim 100, whereinthe condition is one or more of CD4 leukemia, CD8 leukemia, LGLleukemia, systemic lupus erythematosus, Sjögren's syndrome, Wegener'sgranulomatosis, Celiac disease, Hashimoto's thyroiditis, rheumatoidarthritis, diabetes mellitus, psoriasis, multiple sclerosis, uveitis,inflammation of the eye, graft-versus-host disease (GvHD), inflammatorybowel diseases (IBD), ulcerative colitis, Crohn's disease, SystemicLupus Erythematosus, alopecia areata, Adult T-cell Leukemia (ATL),HTLV-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), andother non-neoplastic inflammatory diseases associated with HTLV such asuveitis (HU), arthropathy, pneumopathy, dermatitis, exocrinopathy,myositis, asthma, sinusitis, hay fever, bronchitis, chronic obstructivepulmonary disease (COPD), allergic rhinitis, acute and chronic otitis,and lung fibrosis, acne, hair loss, sunburn, nail maintenance, orappearance of aging.
 102. A composite peptide comprising amino acidsequences of at least two interleukin (IL) protein gamma-c-box D-helixregions, wherein the composite peptide comprises at least hydrocarbonlinker element.